Method for producing soluble recombinant interferon protein without denaturing

ABSTRACT

The present invention relates to the field of recombinant protein production in bacterial hosts. It further relates to extraction of soluble, active recombinant protein from an insoluble fraction without the use of denaturation and without the need for a refolding step. In particular, the present invention relates to a production process for obtaining high levels a soluble recombinant Type 1 interferon protein from a bacterial host.

CLAIM OF PRIORITY

This application claims priority under 35 U.S.C. §119(e) to U.S.application Ser. No. 61/310,671 filed on Mar. 4, 2010. The contents ofU.S. application Ser. No. 61/310,671 are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 11, 2011, isnamed 38194201.txt and is 9,237 bytes in size.

BACKGROUND OF THE INVENTION

Many heterologous recombinant proteins are produced in a misfoldedinsoluble form, called inclusion bodies, when expressed in bacterialsystems. In general, denaturing reagents must be used to solubilize therecombinant protein in the inclusion bodies. The protein must then berenatured, under conditions that have been optimized for the protein toproperly fold. Efforts expended on optimization, as well as the slowrefolding process and lowered process yields, add cost and time to theproduction of a recombinant protein.

Interferons exhibit antiviral, antiproliferative, immunomodulatory, andother activities. Several distinct types of human interferons, includingα, β, and γ, have been distinguished based on, e.g., their anti-viraland anti-proliferative activities. Interferon secretion is induced bysignals, including viruses, double-stranded RNAs, other polynucleotides,antigens, and mitogens. Interferon-β is an example of a protein that hasbeen expressed in recombinant form in bacteria, where it is sequesteredin inclusion bodies.

Human interferon-β 1b is a regulatory polypeptide having a molecularweight of about 22 kDa and consisting of 165 amino acid residues. It canbe produced by many cells in the body, in particular fibroblasts, inresponse to viral infection or exposure to other biologics. It binds toa multimeric cell surface receptor. Productive receptor binding resultsin a cascade of intracellular events leading to the expression ofinterferon-β inducible genes and triggering antiviral, antiproliferativeand immunomodulatory activity.

Interferon-β 1b, specifically, Betaseron (h-IFN-β 1b C17S), has beenused to treat diseases including multiple sclerosis (MS), hepatitis Band C infections, glioma, and melanoma. Interferon-β has beendemonstrated to reduce the number of attacks suffered by patients withrelapsing and remitting MS. Substantial amounts of interferon-β 1b areneeded for therapeutic use. Recombinant interferon-β 1b has beenproduced at low levels in mammalian cells, including human fibroblastsand CHO cells. Animal cell expression is typically hindered by technicaldifficulties including longer process time, easy contamination ofcultures, a requirement for maintaining stringent culturing conditions,and the high cost of culture media. As the glycoprotein component hasbeen found to be generally unnecessary for the activity of interferon β,research has turned to the expression of the recombinant protein in thebacterial expression system, E. coli. As noted, the inclusion bodiesgenerated in E. coli must be solubilized by denaturation, and theinterferon-β refolded. Refolding, which is slow, extends process time,adds cost, and lowers yield. To date, a method for quickly andeconomically producing high levels of soluble recombinant interferon-βin either mammalian or bacterial host cells, without the need fordenaturing and refolding steps, has not been described.

SUMMARY OF THE INVENTION

The present invention relates to the expression of interferon in P.fluorescens and development of a new method to extract active proteinsfrom the fermentation product using mild detergents and without the needfor a refolding process.

In particular, the present invention provides a method for producing arecombinant Type 1 interferon protein, said method comprising expressingthe recombinant interferon protein by culturing a Pseudomonas or E. colihost cell containing an expression construct comprising a codingsequence that has been optimized for expression in the host cell, lysingthe host cell, obtaining an insoluble fraction and a soluble fractionfrom the lysis step, extracting the insoluble fraction by subjecting itto non-denaturing extraction conditions, and obtaining an extract pelletand an extract supernatant from the insoluble fraction, wherein therecombinant protein in the extract supernatant is present in solubleform, active form, or a combination thereof, without being furthersubjected to a renaturing or refolding step.

In embodiments, the non-denaturing extraction conditions comprise thepresence of a mild detergent. In certain embodiments, the mild detergentis a Zwitterionic detergent. In specific embodiments, the Zwitterionicdetergent is Zwittergent 3-08, Zwittergent 3-10, Zwittergent 3-12, orZwittergent 3-14. In embodiments, the non-denaturing extractionconditions comprise about 0.5% to about 2% Zwittergent 3-14. In certainembodiments, the mild detergent is not N-lauroyl-sarcosine (NLS).

In embodiments, the non-denaturing extraction conditions comprise thepresence of a mild detergent and further comprise a chaotropic agent anda cosmotropic salt. In certain embodiments, the chaotropic agent is ureaor guanidinium hydrochloride, and the cosmotropic salt is NaCl, KCl, or(NH4)2SO4. In specific embodiments, the non-denaturing extractionconditions comprise about 0.5 to about 2% Zwittergent 3-14; about 0 toabout 2 M urea; about 0 to about 2 M NaCl; and the pH is about 6.5 toabout 8.5. In embodiments, the non-denaturing extraction conditionscomprise: 1% Zwittergent 3-14; 2 M urea; 2 M NaCl; and the pH is about8.2. In other embodiments, the non-denaturing extraction conditionsadditionally comprise about 1% to about 40% w/v solids. In certainembodiments, the non-denaturing extraction conditions additionallycomprise about 5% w/v solids.

In embodiments, the recombinant Type 1 interferon protein is aninterferon-β, an interferon-α, an interferon-κ, an interferon-τ, or aninterferon-ω. In specific embodiments, the recombinant Type 1 interferonprotein is an interferon-β or an interferon-α. In embodiments, therecombinant Type 1 interferon protein is an interferon-β, and saidinterferon-β is selected from the group consisting of: a humaninterferon-β 1b and human interferon-β 1b C17S. In embodiments, whereinthe recombinant Type 1 interferon is an interferon-α, the interferon-αis selected from the group consisting of: human interferon-α 2a andhuman interferon-α 2b.

In further embodiments the claimed method further comprises measuringthe amount of recombinant Type 1 interferon protein in the insolublefraction and the extract supernatant fractions, wherein the amount ofrecombinant interferon protein detected in the extract supernatantfraction is about 10% to about 95% of the amount of the recombinantinterferon protein detected in the insoluble fraction. In otherembodiments, the method further comprises measuring the activity of therecombinant protein, wherein about 40% to about 100% of the recombinantprotein present in the extract supernatant is determined to be active.In related embodiments, the recombinant protein is an interferon-β, andthe amount of active recombinant protein is determined by Blue Sepharoseaffinity column chromatography, receptor binding assay, antiviralactivity assay, or cytopathic effect assay. In other embodiments, therecombinant protein is an interferon-α, an interferon-κ, or aninterferon-ω, and the amount of active recombinant protein is determinedby Blue Sepharose affinity column chromatography, receptor bindingassay, antiviral activity assay, or cytopathic effect assay.

The invention further includes methods for producing a recombinant Type1 interferon protein, said method comprising expressing the recombinantinterferon protein by culturing a Pseudomonas or E. coli host cellcontaining an expression construct comprising a coding sequence that hasbeen optimized for expression in the host cell, lysing the host cell,obtaining an insoluble fraction and a soluble fraction from the lysisstep, extracting the insoluble fraction by subjecting it tonon-denaturing extraction conditions, and obtaining an extract pelletand an extract supernatant from the insoluble fraction, wherein therecombinant protein in the extract supernatant is present in solubleform, active form, or a combination thereof, without being furthersubjected to a renaturing or refolding step, wherein the recombinantprotein is an interferon-β, and further wherein the non-denaturingextraction conditions are optimized using the information in FIG. 4B.

In embodiments, the recombinant protein in the extract supernatant ispresent at a concentration of about 0.3 grams per liter to about 10grams per liter. In other embodiments, the host cell is cultured in avolume of about 1 to about 20 or more liters. In specific embodiments,the host cell is cultured in a volume of about 1 liter, about 2 liters,about 3 liters, about 4 liters, about 5 liters, about 10 liters, about15 liters, or about 20 liters.

In embodiments of the invention, the expression construct comprises aninducible promoter. In specific embodiments, the expression constructcomprises a lac promoter derivative and expression of the interferon isinduced by IPTG.

In embodiments, the host cell is grown at a temperature of about 25° C.to about 33° C., at a pH of about 5.7 to about 6.5, and the IPTG isadded to a final concentration of about 0.08 mM to about 0.4 mM, whenthe OD575 has reached about 80 to about 160. In specific embodiments,the host cell is grown at a temperature of about 32° C., at a pH ofabout 5.7 to 6.25, and the IPTG is added to a final concentration ofabout 0.2 mM, when the OD575 has reached about 120 to about 160.

In embodiments of the invention, the expression construct comprises ahigh activity ribosome binding site. In certain embodiments, the hostcell is a lon hslUV protease deletion strain. In other embodiments, theType 1 interferon is expressed in the cytoplasm of the host cell. Inrelated embodiments, the Type 1 interferon is human interferon-β 1b orhuman interferon-β 1b C17S, and is expressed in the cytoplasm of thehost cell.

The invention also provides methods for extracting a recombinant Type 1interferon protein, wherein the recombinant interferon protein ispresent in an insoluble fraction, said insoluble fraction produced afterlysis of a Pseudomonas or E. coli host cell expressing the recombinantinterferon protein, said method comprising subjecting the insolublefraction to non-denaturing extraction conditions, and obtaining anextract pellet from the insoluble fraction, said extract pelletcomprising recombinant interferon protein, wherein the recombinantinterferon protein in the extract pellet is in soluble form, activeform, or a combination thereof, without being subjected to a renaturingor refolding step.

In embodiments, the recombinant Type 1 interferon protein extracted isan interferon-β, an interferon-α, an interferon-κ, an interferon-τ, oran interferon-ω. In certain embodiments, the recombinant Type 1interferon protein is an interferon-β or an interferon-α. Inembodiments, the recombinant Type 1 interferon protein is aninterferon-β, and said interferon-β is selected from the groupconsisting of: a human interferon-β 1b and human interferon-β 1b C17S.In other embodiments, the interferon-α is selected from the groupconsisting of: human interferon-α 2a and human interferon-α 2b.

The invention additionally provides a method for producing an insolublefraction comprising a recombinant Type 1 interferon protein, wherein therecombinant interferon protein is expressed in a Pseudomonas or E. colihost cell from a nucleic acid construct comprising a nucleic acidsequence that is operably linked to a lac derivative promoter, saidmethod comprising growing the host cell at a temperature of about 25° C.to about 33° C. and at a pH of about 5.7 to about 6.5, to an OD600 ofabout 80 to about 160, and inducing the host cell at a concentration ofabout 0.08 mM to about 0.4 mM IPTG, lysing the host cell andcentrifuging it to produce the pellet fraction, wherein soluble, active,or soluble and active recombinant interferon protein can be obtained byextracting the pellet fraction under non-denaturing conditions without asubsequent renaturing or refolding step.

In embodiments, the recombinant Type 1 interferon protein comprised bythe insoluble fraction is an interferon-β, an interferon-α, aninterferon-κ, or an interferon-ω. In specific embodiments, therecombinant Type 1 interferon protein is an interferon-β or aninterferon-α. In embodiments, wherein recombinant Type 1 interferonprotein is an interferon-β, the interferon-β is selected from the groupconsisting of: a human interferon-β 1b and human interferon-β 1b C17S.In embodiments, wherein the Type 1 interferon protein is aninterferon-α, the interferon-α is selected from the group consisting of:human interferon-α 2a and human interferon-α 2b. In embodiments, in themethod for producing an insoluble fraction comprising a recombinant Type1 interferon protein, the temperature at which the host cell is grown isabout 32° C., and the IPTG concentration is about 0.2 mM.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and in theaccompanying drawings.

FIG. 1. Initial CGE evaluation of IFN-β recovered from P. fluorescensstrain PS530-001. A. Protein analyzed under reducing conditions. B.Protein analyzed under non-reducing conditions.

For both A and B:

Lane 1. Molecular weight ladder with sizes as indicated.

Lane 2. Pellet from initial centrifugation after cell lysis (insolublefraction).

Lanes 3-5. Supernatant from initial centrifugation after cell lysis(soluble fraction).

Lanes 6-9. Supernatant from centrifugation following extraction step.Lanes 6 and 7 represent extraction with PBS buffer, without and with 1%Zwittergent 3-14, respectively, and Lanes 8 and 9 represent extractionwith acetate buffer, without and with 1% Zwittergent 3-14, respectively.

Lanes 10-13. Pellet from spin following extraction step. Lanes 10 and 11represent extraction with PBS buffer, without and with 1% Zwittergent3-14, respectively, and Lanes 12 and 13 represent extraction withacetate buffer, without and with 1% Zwittergent 3-14, respectively.

FIG. 2. Flowchart of study performed to evaluate extraction ofinterferon-β using different detergents.

FIG. 3. Flowchart of statistically designed study performed to evaluateextraction of interferon-β using different extraction conditionsincluding Zwittergent 3-14.

FIG. 4. Results of study performed to evaluate extraction ofinterferon-β using different extraction conditions including Zwittergent3-14. A. Statistical summary. B. Ranges of useful extraction conditions.

FIG. 5. Insoluble IFN-β Production over Post-Induction Time forReplicate Fermentations.

The results from three different replicates were plotted.

FIG. 6. Insoluble IFN-β Production over Post-Induction Time forAlternate pH and OD.

The results from three different replicates were plotted.

FIG. 7. IFN-β 1b Sequence A. IFN-β 1b C17S Amino Acid Sequence. (SEQ IDNO: 1) The sequence shows the N-terminal methionine, which is notpresent in the purified protein. B. IFN-β DNA Sequence with CodonsOptimized for P. fluorescens. This sequence encodes the amino acidsequence shown in FIG. 7A. (SEQ ID NO: 2) C. IFN-β 1b C17S Amino AcidSequence, without N-terminal methionine. (SEQ ID NO: 3)

FIG. 8. IFN-α 2a Sequence. A. IFN-α 2a Amino Acid Sequence. (SEQ ID NO:4) B. IFN-α 2a DNA Sequence with Codons Optimized for P. fluorescens.(SEQ ID NO: 5)

FIG. 9. IFN-α 2b Sequence. A. IFN-α 2b Amino Acid Sequence. (SEQ ID NO:6) B. IFN-α 2b DNA Sequence with Codons Optimized for P. fluorescens.(SEQ ID NO: 7)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing large amounts ofsoluble recombinant interferon protein in a Pseudomonas expressionsystem. In particular, this method eliminates the need for thedenaturing step and subsequent renaturing/refolding step typicallyrequired.

Production of recombinant interferon-β in bacterial expression systemshas been hampered by sequestration of the protein in insoluble inclusionbodies. Solubilization of the inclusion bodies requires denaturation,which in turn necessitates the use of a refolding step that is costly,time-consuming, and decreases protein yield. The present inventioncircumvents the need for a refolding step by providing methods forproducing and solubilizing interferon without recourse to denaturation.

Methods for producing a recombinant interferon protein that is soluble,active, or both, in a bacterial expression system, without subjectingthe protein to a denaturing step, are provided. In particular, anon-denaturing extraction process that results in soluble interferonprotein is described. Interferons expressed in bacterial expressionsystems are generally localized to an insoluble fraction. In theextraction process of the present invention, this insoluble fraction issubjected to extraction conditions that include non-denaturingconcentrations of mild detergents and produce soluble protein.

Also provided by the present invention are methods for producing arecombinant interferon protein wherein growth conditions for thePseudomonas host cell are optimized to maximize yields of the solublerecombinant interferon protein, particularly when the extraction methodof the invention is used. Studies of the effect of E. coli growthconditions on soluble protein production have been reported. Thesolubility of a given protein when expressed in Pseudomonas can bedifferent from that in E. coli. This is illustrated in, e.g., U.S. Pat.App. Pub. No. 2006/0040352, “Expression of Mammalian Proteins inPseudomonas Fluorescens,” which shows side-by-side comparisons of thesoluble amounts of several proteins produced using E. coli or P.fluorescens as the host. Furthermore, there is substantial variationamong the solubilities of different proteins even in the same host, assolubility is influenced strongly by protein structure, e.g., amino acidsequence. Previously reported attempts at producing IFN-β in E. coliresulted in protein that required refolding. See, e.g., Russell-Harde,1995, “The Use of Zwittergent 3-14 in the Purification of RecombinantHuman Interferon-β Ser17 (Betaseron) et al., J. Interferon and CytokineRes. 15:31-37, and Ghane, et al., 2008, “Over Expression of BiologicallyActive Interferon Beta Using Synthetic Gene in E. coli,” J. of Sciences,Islamic Republic of Iran 19(3):203-209, both incorporated herein byreference.

The methods further provide optimized growth conditions including growthtemperature, OD at time of promoter induction, inducer concentration,and pH. Extraction conditions provided include detergent type andconcentration, chaotropic agent, cosmotropic salt, and pH. Specificvalues as well as optimal parameter ranges are provided. Also providedare methods for optimizing extraction conditions using the providedranges.

Bacterial Growth Conditions

In embodiments of the present invention, the bacterial growth conditionsare optimized to increase the amount of soluble interferon proteinobtained using the extraction methods as provided herein. Use of thegrowth conditions of the present invention with other extractionconditions, e.g., other methods described and used in the art, is alsocontemplated.

Optimal growth conditions particularly useful in conjunction with theextraction methods of the invention comprise: a temperature of about 25°C. to about 33° C.; a pH of about 5.7 to about 6.5, and induction withabout 0.08 mM to about 0.4 mM IPTG when the culture has reached an OD₅₇₅of about 80 to about 160.

The pH of the culture can be maintained using pH buffers and methodsknown to those of skill in the art. Control of pH during culturing alsocan be achieved using aqueous ammonia. In embodiments, the pH of theculture is about 5.7 to about 6.5. In certain embodiments, the pH is5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4. or 6.5. In other embodiments,the pH is 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, or6.2 to 6.5. In yet other embodiments, the pH is 5.7 to 6.0, 5.8 to 6.1,5.9 to 6.2, 6.0 to 6.3, 6.1 to 6.4, or 6.2 to 6.5. In certainembodiments, the pH is about 5.7 to about 6.25.

In embodiments, the growth temperature is maintained at about 25° C. toabout 33° C. In certain embodiments, the growth temperature is about 25°C., about 26° C., about 27° C., about 28° C., about 29° C., about 30°C., about 31° C., about 32° C., or about 33° C. In other embodiments,the growth temperature is maintained at about 25° C. to about 27° C.,about 25° C. to about 28° C., about 25° C. to about 29° C., about 25° C.to about 30° C., about 25° C. to about 31° C., about 25° C. to about 32°C., about 25° C. to about 33° C., about 26° C. to about 28° C., about26° C. to about 29° C., about 26° C. to about 30° C., about 26° C. toabout 31° C., about 26° C. to about 32° C., about 27° C. to about 29°C., about 27° C. to about 30° C., about 27° C. to about 31° C., about27° C. to about 32° C., about 26° C. to about 33° C., about 28° C. toabout 30° C., about 28° C. to about 31° C., about 28° C. to about 32°C., about 29° C. to about 31° C., about 29° C. to about 32° C., about29° C. to about 33° C., about 30° C. to about 32° C., about 30° C. toabout 33° C., about 31° C. to about 33° C., about 31° C. to about 32°C., about 30° C. to about 33° C., or about 32° C. to about 33° C.

Induction

As described elsewhere herein, inducible promoters can be used in theexpression construct to control expression of the recombinant interferongene. In the case of the lac promoter derivatives or family members,e.g., the tac promoter, the effector compound is an inducer, such as agratuitous inducer like IPTG (isopropyl-β-D-1-thiogalactopyranoside,also called “isopropylthiogalactoside”). In embodiments, a lac promoterderivative is used, and interferon expression is induced by the additionof IPTG to a final concentration of about 0.08 mM to about 0.4 mM, whenthe cell density has reached a level identified by an OD₅₇₅ of about 80to about 160.

In embodiments, the OD₅₇₅ at the time of culture induction about 80,about 90, about 100, about 110, about 120, about 130, about 140, about150, about 160, about 170 or about 180. In other embodiments, the OD₅₇₅is about 80 to about 100, about 100 to about 120, about 120 to about140, about 140 to about 160. In other embodiments, the OD₅₇₅ is about 80to about 120, about 100 to about 140, or about 120 to about 160. Inother embodiments, the OD₅₇₅ is about 80 to about 140, or about 100 to160. The cell density can be measured by other methods and expressed inother units, e.g., in cells per unit volume. For example, an OD₅₇₅ ofabout 80 to about 160 of a Pseudomonas fluorescens culture is equivalentto approximately 8×10¹⁰ to about 1.6×10¹¹ colony forming units per mL or35 to 70 g/L dry cell weight. In embodiments, the cell density at thetime of culture induction is equivalent to the cell density as specifiedherein by the absorbance at OD₅₇₅, regardless of the method used fordetermining cell density or the units of measurement. One of skill inthe art will know how to make the appropriate conversion for any cellculture.

In embodiments, the final IPTG concentration of the culture is about0.08 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, or about 0.4 mM. Inother embodiments, the final IPTG concentration of the culture is about0.08 mM to about 0.1 mM, about 0.1 mM to about 0.2 mM, about 0.2 mM toabout 0.3 mM, about 0.3 mM to about 0.4 mM, about 0.2 mM to about 0.4mM, or about 0.08 to about 0.2 mM.

In embodiments, the interferon is expressed by induction of a lacpromoter or derivative using IPTG, lactose or allolactose, by methodsknown in the art and described in the literature, e.g., in U.S. Pat. No.7,759,109, “High density growth of T7 expression strains withauto-induction option,” incorporated herein by reference in itsentirety.

In embodiments wherein a non-lac type promoter is used, as describedherein and in the literature, other inducers or effectors can be used.

After induction is started, cultures are grown for a period of time,typically about 24 hours, during which time the recombinant interferonprotein is expressed. Cell cultures can be concentrated bycentrifugation, and the culture pellet resuspended in a buffer orsolution appropriate for the subsequent lysis procedure.

In embodiments, cells are disrupted using equipment for high pressuremechanical cell disruption (which are available commercially, e.g.,Microfluidics Microfluidizer, Constant Cell Disruptor, Niro-Soavihomogenizer or APV-Gaulin homogenizer). Any appropriate method known inthe art for lysing cells can be used to release the insoluble fraction.For example, in embodiments, chemical and/or enzymatic cell lysisreagents, such as cell-wall lytic enzyme and EDTA, can be used. Use offrozen or previously stored cultures is also contemplated in the methodsof the invention. Cultures can be OD-normalized prior to lysis.

Centrifugation is performed using any appropriate equipment and method.Centrifugation of cell culture or lysate for the purposes of separatinga soluble fraction from an insoluble fraction is well-known in the art.For example, lysed cells can be centrifuged at 20,800×g for 20 minutes(at 4° C.), and the supernatants removed using manual or automatedliquid handling. The pellet (insoluble) fraction is resuspended in abuffered solution, e.g., phosphate buffered saline (PBS), pH 7.4.Resuspension can be carried out using, e.g., equipment such as impellersconnected to an overhead mixer, magnetic stir-bars, rocking shakers,etc.

A “soluble fraction,” i.e., the soluble supernatant obtained aftercentrifugation of a lysate, and an “insoluble fraction,” i.e., thepellet obtained after centrifugation of a lysate, result from lysing andcentrifuging the cultures. These two fractions also can be referred toas a “first soluble fraction” and a “first insoluble fraction,”respectively.

It is possible to obtain soluble IFN-β using extraction methodsaccording to the invention, from expression cultures prepared by growingcultures under conditions in which the pH and the induction OD are nottightly controlled (see, e.g., Example 2). Optimization of the growthconditions as described herein results in substantially increasedproduction of soluble IFN-|3.

Non-Denaturing Extraction Process

It has been discovered that high levels of soluble interferon proteincan be obtained from the insoluble fraction, using non-denaturingextraction methods of the present invention.

Non-denaturing extraction conditions identified as particularly usefulfor producing high levels of soluble recombinant interferon proteincomprise: a mild detergent at a non-denaturing concentration, e.g.,Zwittergent 3-14 (0.5 to 2% w/v); a chaotropic agent, e.g., urea (0-2M),and a cosmotropic salt, e.g., NaCl (0-2M), at a buffer pH of 6.5 to 8.5and a solids concentration of 5-20% w/v.

After obtaining the soluble fraction and insoluble fraction, asdescribed above, the soluble recombinant interferon protein is extractedfrom the insoluble fraction by incubating the resuspended insolublefraction under the non-denaturing extraction conditions describedherein. After incubation, the extracted mixture is centrifuged toproduce an “extract supernatant” (the soluble supernatant fractionobtained after extraction containing solubilized recombinant protein)and an “extract pellet” (the insoluble pellet fraction obtained afterextraction). These fractions can also be referred to as the “secondsoluble fraction” and the “second insoluble fraction.”

Extraction Conditions

Many different parameters for the extraction conditions were evaluatedfor their effect on the amount of soluble protein observed in theextract supernatant, as described in Example 3 herein. It was found thatsoluble interferon protein was observed when the extraction conditionscomprised any of a number of different detergents, at varyingconcentrations, as well as when other parameters were varied. However,certain parameters had a particularly striking effect on the amount ofsoluble protein produced.

Specifically, extraction conditions comprising Zwitterionic detergents(Zwittergents) gave the best soluble protein yields. In particular, useof the Zwitterionic detergents, Zwittergent 3-08, Zwittergent 3-10,Zwittergent 3-12, and especially Zwittergent 3-14, resulted in thehighest yields. N-Lauroylsarcosine (NLS) gave a notably high yield,however the soluble protein obtained was found to be inactive based onan affinity assay (Sepharose blue affinity column binding). Therefore,the term “mild detergents” as used herein is intended not to includeN-lauroylsarcosine.

The detergents were tested at non-denaturing concentrations. It wasfound that a concentration of Zwittergent 3-14(3-(N,N-dimethylmyristylammonio) propanesulfonate) of at least 0.5%(w/v), and preferably 1%, well above its critical micelle concentration(which is 0.01%) provides the most efficient extraction of solubleinterferon protein.

Therefore, use of non-denaturing concentrations of mild detergents,particularly Zwitterionic detergents, more particularly Zwittergent3-08, Zwittergent 3-10, Zwittergent 3-12, and Zwittergent 3-14, moreparticularly Zwittergent 3-14, and not NLS, is contemplated for use inthe extraction conditions of the invention.

In other embodiments of the invention, the non-denaturing extractionconditions comprise a concentration of about 0.5% to about 2% (w/v)Zwittergent 3-14. In embodiments, the w/v concentration of Zwittergent3-14 is about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% toabout 2%, or about 1% to about 2%. In certain embodiments, the w/vconcentration of Zwittergent 3-14 is about 0.5%, about 0.6%, about 0.7%,about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%,about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%,or about 2.0%.

In other embodiments of the invention, non-denaturing extractionconditions comprise a concentration of about 0.5% to about 2% (w/v)Zwittergent 3-08, Zwittergent 3-10, or Zwittergent 3-12. In embodiments,the w/v concentration of Zwittergent 3-08, Zwittergent 3-10, orZwittergent 3-12 is about 0.5% to about 1%, about 1% to about 1.5%,about 1.5% to about 2%, or about 1% to about 2%. In certain embodiments,the w/v concentration of Zwittergent 3-08, Zwittergent 3-10, orZwittergent 3-12 is about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%,about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about2.0%.

A mild detergent does not cause protein unfolding at low levels, e.g.,2% or less. For example, SDS and NLS are typically considered strongerdetergents, as they can denature proteins at these levels. Anon-denaturing concentration indicates a concentration of a reagent atwhich proteins are not denatured. Proteins that are not denatured duringprocessing do not require refolding.

In embodiments, non-denaturing extraction conditions of the presentinvention comprise about 0.5 to about 2% Zwittergent 3-14; about 0 toabout 2 M urea; about 0 to about 2 M NaCl; and wherein the pH is about6.5 to about 8.5.

The following table lists examples of common detergents, includingionic, non-ionic, and zwitter-ionic detergents, and their properties. Animportant characteristic of a detergent is its critical micelleconcentration (CMC), which relates to its protein solubilizationcapability as well as the ease of subsequent removal of detergents fromprotein solutions.

TABLE 1 Examples of Detergents Monomer, Micelle, CMC CMC Detergent MW DaMW Da % (w/v) mM Zwittergent 364 30,000 0.004-0.015 0.1-0.4 3-14 (0.011)(0.3) Tween-20 1228 0.007 0.059 Tween-80 1310 76,000 0.0016 0.012 TritonX- 650 90,000 0.013-0.06 0.2-0.9 100 (0.021) (0.3) Sodium 432 4,200 0.215 Deoxy- cholate Sodium 293 600 0.4 13.7 Lauroyl- sarcosine NDSB 195 N/AN/A N/A NP-40 617 90000 0.003-0.018, 0.05-0.3 CHAPS 615 6,000 0.37-0.62  6-10 Octyl-β- 292 8,000 0.73 23 gluco- pyranoside

TABLE 2 Physical Properties of Zwitterionic Detergents Monomer, Micelle,CMC CMC Detergent MW Da MW Da % (w/v) mM Zwittergent 3-08 280 — 9.2 330Zwittergent 3-10 308 12,500 0.77-1.23 25-40 Zwittergent 3-12 336 18,5000.067-0.134 2-4 Zwittergent 3-14 364 30,000 0.004-0.015 0.1-0.4 (0.011)(0.3) Zwittergent 3-16 392 60,000 0.0004-0.0024 0.01-0.06

It was further observed that when the non-denaturing extractionconditions comprised the combination of a chaotropic agent, urea, acosmotropic salt, NaCl, Zwittergent 3-14, and an appropriate bufferrange, the extraction yield was increased several-fold compared to theuse of Zwittergent 3-14 alone (see Example 3).

TABLE 3 Selected concentration ranges of extraction componentsPermissible Conc. Selected Component Range Conc. Zwittergent 0.5-2%(w/v) 1% 3-14 Urea 0-2M 2M NaCl 0-2M 2M Solid Conc.  5-20% (w/v) 5%Buffer pH 6.5-8.5 8.2

Chaotropic agents disrupt the 3-dimensional structure of a protein ornucleic acid, causing denaturation. In embodiments, the non-denaturingextraction conditions comprise low, non-denaturing concentrations ofchaotropic agents, e.g., urea or guanidinium hydrochloride. Inembodiments, the non-denaturing extraction conditions comprise urea at aconcentration of about 0.5M to about 2M or higher. We observed that 6Murea denatures IFN-β. Typically, non-denaturing concentrations of ureaor guanidinium hydrochloride are below 3M. In embodiments, thenon-denaturing extraction conditions comprise urea at a concentration ofabout 0.5 to about 1M, about 1 to about 1.5M, about 1.5 to about 2M,about 1 to about 2M, about 0.5M, about 0.6M, about 0.7M, about 0.8M,about 0.9M, about 1.0M, about 1.1M, about 1.2M, about 1.3M, about 1.4M,about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, or about2.0M. In other embodiments, the extraction conditions compriseguanidinium hydrochloride at a concentration of 0.5 to 2M. Inembodiments, extraction conditions comprise guanidinium hydrochloride ata concentration of 0.5 to 1M, 1 to 1.5M, 1.5 to 2M, 1 to 2M, 0.5M, about0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M, about1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about1.8M, about 1.9M, or about 2.0M.

Cosmotropic salts contribute to the stability and structure ofwater-water interactions. Cosmotropes cause water molecules to favorablyinteract, which also stabilizes intermolecular interactions inmacromolecules such as proteins. Any such appropriate agent, as known inthe art, can be used in the extraction conditions of the presentinvention. In embodiments, the non-denaturing extraction conditionscomprise a cosmotropic salt selected from NaCl, KCl, and (NH₄)₂SO₄. Incertain embodiments, NaCl is present at a concentration of about 0.15Mto about 2M. In embodiments, NaCl is present in the extractionconditions at a concentration of about 0.15 to about 0.5M, about 0.5 toabout 0.75M, about 0.75M to about 1M, about 1M to about 1.25M, about1.25M to about 1.5M, about 1.5M to about 1.75M, about 1.75M to about 2M,about 0.15M to about 1M, about 1M to about 1.5M, about 1.5M to about 2M,about 1M to about 2M, about 0.15M, about 0.25M, about 0.5M, about 0.6M,about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M, about 1.2M,about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M,about 1.85M, about 1.9M, or about 2.0M.

In other embodiments, KCl is present in the non-denaturing extractionconditions at a concentration of about 0.15 to about 0.5M, about 0.5 toabout 0.75M, about 0.75M to about 1M, about 1M to about 1.25M, about1.25M to about 1.5M, about 1.5M to about 1.75M, about 1.75M to about 2M,about 0.15M to about 1M, about 1M to about 1.5M, about 1.5M to about 2M,about 1M to about 2M, about 0.15M, about 0.25M, about 0.5M, about 0.6M,about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M, about 1.2M,about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M,about 1.85M, about 1.9M, or about 2.0M.

In other embodiments, (NH₄)₂SO₄ is present in the non-denaturingextraction conditions at a concentration of about 0.15 to about 0.5M,about 0.5 to about 0.75M, about 0.75M to about 1M, about 1M to about1.25M, about 1.25M to about 1.5M, about 1.5M to about 1.75M, about 1.75Mto about 2M, about 0.15M to about 1M, about 1M to about 1.5M, about 1.5Mto about 2M, about 1M to about 2M, about 0.15M, about 0.25M, about 0.5M,about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M,about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M,about 1.8M, about 1.85M, about 1.9M, or about 2.0M.

The extraction conditions were found to be most effective when the pHwas maintained within a range of 6.5 to 8.5. Useful pH buffers are thoserecommended in standard protein purification texts (e.g., ProteinPurification: Principles and Practice, by Robert Scopes (Springer, 1993)can be used here, including Tris, Bis-Tris, phosphate, citrate, acetate,glycine, diethanolamine, 2-amino-2-methyl-1,3-propanediol,triethanolamine, imidazole, histidine, pyridine, etc. Many buffers havebeen described in the literature and are commercially available. Inembodiments, the pH of the non-denaturing extraction conditions is about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.8, about7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about8.5. In other embodiments, the pH is about 6.5 to about 6.8, about 6.6to about 6.9, about 6.7 to about 7.0, about 6.8 to about 7.1, about 6.9to about 7.2, about 7.0 to about 7.3, about 7.1 to about 7.4, about 7.2to about 7.5, about 7.3 to about 7.6, about 7.4 to about 7.7, about 7.5to about 7.8, about 7.6 to about 7.9, about 7.8 to about 8.1, about 7.9to about 8.2, about 8.0 to about 8.3, about 8.1 to about 8.4 or about8.2 to about 8.5. In other embodiments, the pH is about 6.5 to about7.0, about 7.0 to about 7.5, or about 7.5 to about 8.0.

The solids concentration in the non-denaturing extraction conditions wasalso varied. This parameter represents the amount of solid material inthe extract incubation mixture. Solids concentration can be determinedby weighing the wet pellet (i.e., the insoluble fraction), and comparingthis weight with the total weight of the extraction mixture. Specificsolids concentrations are achieved by concentrating or diluting theinsoluble fraction. High extraction yields were observed across a rangeof solids concentrations of 5% to 40% (w/v). In embodiments of theinvention, the solids in the non-denaturing extraction conditions arepresent at a w/v concentration of about 5%, about 7.5%, about 10%, about12.5%, about 15%, about 17.5%, about 20%, about 22.5%, about 25%, about27.5%, about 30%, about 32.5%, about 35%, about 37.5%, or about 40%. Inother embodiments of the invention, the solids in the non-denaturingextraction conditions are present at a w/v concentration of about 5% toabout 7.5%, about 7.5% to about 10%, about 10% to about 12.5%, about12.5% to about 15%, about 15% to about 17.5%, about 17.5% to about 20%,about 20% to about 22.5%, about 22.5% to about 25%, about 25% to about27.5%, about 27.5% to about 30%, about 30% to about 32.5%, about 32.5%to about 35%, about 35% to about 37.5%, about 37.5% to about 40%, about5% to about 10%, about 10% to about 15%, about 15% to about 20%, about20% to about 25%, about 25% to about 30%, about 35% to about 40%, about5% to about 15%, about 5% to about 25%, about 5% to about 30%, about 5%to about 35%, about 10% to about 20%, about 20% to about 30%, about 30%to about 40%, about 5% to about 20%, or about 20% to about 40%.

In embodiments, the extraction methods of the invention are combinedwith simultaneous enrichment techniques such as adsorption to furtherenhance protein yield.

The solubilized protein can be isolated or purified from other proteinand cellular debris by, for example, centrifugation and/orchromatography such as size exclusion, anion or cation exchange,hydrophobic interaction, or affinity chromatography.

Interferons

Human interferons have been classified into three major types.Interferon type I: Type I IFNs bind to a specific cell surface receptorcomplex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 andIFNAR2 chains. Human type I interferons include are IFN-α, IFN-β, IFN-κ,and IFN-ω and IFN-ε. Interferon type II: Type II IFNs (human IFN-γ)binds to IFNGR. Interferon type III: type III interferons signal througha receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1(also called CRF2-12).

Human Type I interferon appears to bind to two-receptor subunits,IFNAR-1 and -2, which are widely distributed on the cell surface ofvarious cell types. Ligand involvement leads to the induction of thephosphorylation of tyrosine kinases TYK2 and JAK-1, which are coupled toIFNAR-1 and -2 respectively. Once phosphorylated, STAT proteins arereleased from the receptor and form homodimers as well as heterodimers.Once released, the dimers of STAT associate with interferon ResponsiveFactor 9 (IRF-9), a DNA binding protein, forming a complex calledIFN-stimulated gene factor-3 (ISGF-3), that migrates into the nucleus.Next, the ISGF-3 complex binds to a DNA element existing in the upstreamof all IFN inducible genes. Type 1 interferons are described extensivelyin the literature, e.g., in U.S. Pat. No. 7,625,555, “Recombinant humaninterferon-like proteins, incorporated herein by reference.”

Type 1 IFNs have substantial structural similarity, as evidenced bytheir sequences and their shared receptor binding capacity. According toKontsek, P., 1994, “Human type I interferons: structure and function,”Acta Virol. 38(6):345-60, incorporated by reference herein, human type Iinterferons (13 had been reported at the time) have a 20% overallsequence homology, which determines identical secondary and tertiaryfolding of polypeptides. Further, Kontsek reports that three-dimensionalmodels suggest that the globular structure of type I IFNs consists of abundle of 5 α-helices, which might form two polypeptide domains.Disulfide bond Cys 29-Cys 139 stabilizes both domains in a bioactiveconfiguration. Two conservative hydrophilic regions associated with theamino acids (aa) 30-41 and 120-145 are thought to constitute the basicframework of receptor recognition site in type I IFNs, and the differentspectra of biological effects among human type I IFNs are speculated tobe due to subtle sequential heterogeneity in the segments aa 30-41 and120-145, and the variable hydrophilic aa regions 23-26, 68-85 and112-121. A later report by Oritani, et al., 2001, “Type I interferonsand limitin: a comparison of structures, receptors, and functions,”Cytokine Growth Factor Rev 12(4):337-48, incorporated by referenceherein, describes family members IFN-α, IFN-β, IFN-pi, and IFN-tau. Thepaper also reports that IFN-α and IFN-β have a globular structurecomposed of five a-helices, and discusses comparative sequence analyses,a prototypic three-dimensional structure, analysis with monoclonalantibodies, and construction of hybrid molecules and site directedmutagenesis.

Production of any Type 1 interferon protein using the methods of thepresent invention is contemplated. Type 1 interferon proteins that canbe produced using the methods of the invention, include, but not limitedto, human IFN-α 2a and 2b, human IFN-β 1b, human IFN-κ (e.g.,NM_(—)020124, AAK63835, and described by LaFleur, et al., 2001,“Interferon-kappa, a novel type I interferon expressed in humankeratinocytes,” J. Biol. Chem. 276 (43), 39765-39771, incorporatedherein by reference), and human IFN-w (e.g., NM_(—)002177, NP_(—)002168,and described in U.S. Pat. No. 7,470,675, “Methods for treating cancerusing interferon-ω-expressing polynucleotides,” incorporated byreference herein in its entirety). Production of IFN-τ using the methodsof the invention is also contemplated. Amino acid and nucleic acidsequences are publicly available, e.g., from GenBank.

Fourteen subtypes of IFN-α proteins have been described: IFNA1, IFNA2,IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16,IFNA17, IFNA21. IFN-α is made synthetically as therapeutic agent, aspegylated interferon alfa-2a and pegylated interferon alfa-2b.

IFN-β (IFNB1, or IFN-β 1b) is the main β subtype (see, e.g., GenBankNP002167.1, which provides the mature peptide sequence). Betaseron is ananalogue of human IFN-β in which serine was genetically engineered tosubstitute for cysteine at position 17, is known as IFN-β 1b C17S(described in U.S. Pat. No. 4,588,585, “Human recombinant cysteinedepleted interferon-β muteins,” incorporated herein by reference). Themolecule is a small polypeptide of 165 amino acids with a singledisulphide bond, and is produced as a non-glycosylated protein.

IFN-τ is described, and sequences of IFN-τ disclosed, e.g., in U.S. Pat.No. 7,214,367, “Orally-administered interferon-tau compositions andmethods,” incorporated herein by reference in its entirety.

A number of Type 1 IFNs have been approved by the FDA for use intreating disease in humans. The following table lists examples of Type 1interferon drugs. In embodiments of the invention, any of these drugsare produced using the methods as claimed or described herein.

TABLE 4 Examples of Type 1 interferon drugs. Generic name Trade nameInterferon α 2a Roferon A PEGylated interferon α 2a Pegasys PEGylatedinterferon α 2a Reiferon Retard Interferon α 2b Intron A/ReliferonPEGylated interferon α 2b PegIntron Human leukocyte Interferon-αMultiferon (HuIFN-α-Le) Interferon β 1a, liquid form Rebif Interferon β1a lyophilized Avonex Interferon β 1b Betaseron/Betaferon

In embodiments, variants and modifications of Type 1 interferon proteinsare produced using the methods of the present invention. For example,IFN-β variants are described in U.S. Pat. No. 6,531,122 “Interferon-βvariants and conjugates,” and U.S. Pat. No. 7,625,555, both incorporatedby reference herein. Conjugates include pegylated Type 1 interferons,e.g., the PEGylated agents shown in Table 4, and interferons linked tonon-peptide moieties.

The methods of the invention are expected to be useful for all Type 1interferons, due to their structural similarities. Certain structurallyunrelated proteins, for example, human GCSF, have been found poorcandidates for producing using the methods of the present invention.When GCSF was produced and extracted using methods as described herein,less than 5% of the amount of GCSF protein in the insoluble fraction wasextracted as soluble protein (data not shown).

In general, with respect to an amino acid sequence, the term“modification” includes substitutions, insertions, elongations,deletions, and derivatizations alone or in combination. In someembodiments, the peptides may include one or more modifications of a“non-essential” amino acid residue. In this context, a “non-essential”amino acid residue is a residue that can be altered, e.g., deleted orsubstituted, in the novel amino acid sequence without abolishing orsubstantially reducing the activity (e.g., the agonist activity) of thepeptide (e.g., the analog peptide). In some embodiments, the peptidesmay include one or more modifications of an “essential” amino acidresidue. In this context, an “essential” amino acid residue is a residuethat when altered, e.g., deleted or substituted, in the novel amino acidsequence the activity of the reference peptide is substantially reducedor abolished. In such embodiments where an essential amino acid residueis altered, the modified peptide may possess an activity of a Type 1interferon of interest in the methods provided. The substitutions,insertions and deletions may be at the N-terminal or C-terminal end, ormay be at internal portions of the protein. By way of example, theprotein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moresubstitutions, both in a consecutive manner or spaced throughout thepeptide molecule. Alone or in combination with the substitutions, thepeptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions,again either in consecutive manner or spaced throughout the peptidemolecule. The peptide, alone or in combination with the substitutionsand/or insertions, may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore deletions, again either in consecutive manner or spaced throughoutthe peptide molecule. The peptide, alone or in combination with thesubstitutions, insertions and/or deletions, may also include 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more amino acid additions.

Substitutions include conservative amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain, or physicochemical characteristics (e.g., electrostatic, hydrogenbonding, isosteric, hydrophobic features). The amino acids may benaturally occurring or normatural (unnatural). Families of amino acidresidues having similar side chains are known in the art. These familiesinclude amino acids with basic side chains (e.g. lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan), β-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Substitutions may also include non-conservativechanges.

Methods for Selecting Optimal Extraction Conditions

In embodiments of the present invention, the results of the statisticalanalysis as set forth in FIG. 4B are used to further optimize extractionconditions within the ranges of parameter values provided. High levelsoluble protein production of all Type 1 interferons is expected to beobserved when practicing the invention using any values within theranges set forth. Nonetheless, it is within the capacity of one of skillin the art to utilize the tool represented by FIG. 4B to optimize theextraction conditions to fit the need at hand.

Evaluation of Product Protein Yield

Protein yield in the insoluble and soluble fractions as described hereincan be determined by methods known to those of skill in the art, forexample, by capillary gel electrophoresis (CGE), and Western blotanalysis.

Useful measures of protein yield include, e.g., the amount ofrecombinant protein per culture volume (e.g., grams or milligrams ofprotein/liter of culture), percent or fraction of recombinant proteinmeasured in the insoluble pellet obtained after lysis (e.g., amount ofrecombinant protein in extract supernatant/amount of protein ininsoluble fraction), percent or fraction of active protein (e.g., amountof active protein/amount protein used in the assay), percent or fractionof total cell protein (tcp), amount of protein/cell, and percent drybiomass. In embodiments, the measure of protein yield as describedherein is based on the amount of soluble protein or the amount of activeprotein, or both, obtained.

In embodiments, the methods of the present invention can be used toobtain an extracted recombinant protein yield of about 0.3 grams perliter to about 10 grams per liter. In certain embodiments, the extractedrecombinant protein yield is about 0.3 grams per liter to about 1 gramper liter, about 1 gram per liter to about 2 grams per liter, about 2grams per liter to about 3 grams per liter, about 3 grams per liter toabout 4 grams per liter, about 4 grams per liter to about 5 grams perliter, about 5 grams per liter to about 6 grams per liter, about 6 gramsper liter to about 7 grams per liter, about 7 grams per liter to about 8grams per liter, about 8 grams per liter to about 9 grams per liter, orabout 9 grams per liter to about 10 grams per liter. In embodiments, theextracted protein yield is about 1 gram per liter to about 3 grams perliter, about 2 grams per liter to about 4 grams per liter, about 3 gramsper liter to about 5 grams per liter, about 4 grams per liter to about 6grams per liter, about 5 grams per liter to about 7 grams per liter,about 6 grams per liter to about 8 grams per liter, about 7 grams perliter to about 9 grams per liter, or about 8 grams per liter to about 10grams per liter. In embodiments, the extracted protein yield is about0.5 grams per liter to about 4 grams per liter, 1 gram per liter toabout 5 grams per liter, 2 grams per liter to about 6 grams per liter,about 3 grams per liter to about 7 grams per liter, about 4 grams perliter to about 8 grams per liter, about 5 grams per liter to about 9grams per liter, or about 6 grams per liter to about 10 grams per liter.In embodiments, the extracted protein yield is about 0.5 gram per literto about 5 grams per liter, about 1 grams per liter to about 6 grams perliter, about 2 grams per liter to about 7 grams per liter, about 3 gramsper liter to about 8 grams per liter, about 4 grams per liter to about 9grams per liter, or about 5 grams per liter to about 10 grams per liter.

In embodiments, the amount of recombinant interferon protein detected inthe extracted supernatant fraction is about 10% to about 95% of theamount of the recombinant interferon protein detected in the insolublefraction. In embodiments, this amount is about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, or about 95%. In embodiments, this amount is about 10%to about 20%, 20% to about 50%, about 25% to about 50%, about 25% toabout 50%, about 25% to about 95%, about 30% to about 50%, about 30% toabout 40%, about 30% to about 60%, about 30% to about 70%, about 35% toabout 50%, about 35% to about 70%, about 35% to about 75%, about 35% toabout 95%, about 40% to about 50%, about 40% to about 95%, about 50% toabout 75%, about 50% to about 95%, or about 70% to about 95%.

The protein yield measured in the unextracted insoluble fraction istypically higher than that in the extract supernatant, as material islost during the extraction procedure. Yields from fermentation culturesare typically higher than those from smaller HTP cultures.

“Percent total cell protein” is the amount of protein or polypeptide inthe host cell as a percentage of aggregate cellular protein. Thedetermination of the percent total cell protein is well known in theart.

In embodiments, the amount of interferon protein detected in theextracted supernatant fraction produced is about 1% to about 75% of thetotal cell protein. In certain embodiments, the recombinant proteinproduced is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 1% to about 5%, about 1% to about 10%, about 1% to about 20%,about 1% to about 30%, about 1% to about 40%, about 1% to about 50%,about 1% to about 60%, about 1% to about 75%, about 2% to about 5%,about 2% to about 10%, about 2% to about 20%, about 2% to about 30%,about 2% to about 40%, about 2% to about 50%, about 2% to about 60%,about 2% to about 75%, about 3% to about 5%, about 3% to about 10%,about 3% to about 20%, about 3% to about 30%, about 3% to about 40%,about 3% to about 50%, about 3% to about 60%, about 3% to about 75%,about 4% to about 10%, about 4% to about 20%, about 4% to about 30%,about 4% to about 40%, about 4% to about 50%, about 4% to about 60%,about 4% to about 75%, about 5% to about 10%, about 5% to about 20%,about 5% to about 30%, about 5% to about 40%, about 5% to about 50%,about 5% to about 60%, about 5% to about 75%, about 10% to about 20%,about 10% to about 30%, about 10% to about 40%, about 10% to about 50%,about 10% to about 60%, about 10% to about 75%, about 20% to about 30%,about 20% to about 40%, about 20% to about 50%, about 20% to about 60%,about 20% to about 75%, about 30% to about 40%, about 30% to about 50%,about 30% to about 60%, about 30% to about 75%, about 40% to about 50%,about 40% to about 60%, about 40% to about 75%, about 50% to about 60%,about 50% to about 75%, about 60% to about 75%, or about 70% to about75%, of the total cell protein.

Solubility and Activity

The “solubility” and “activity” of a protein, though related qualities,are generally determined by different means. Solubility of a protein,particularly a hydrophobic protein, indicates that hydrophobic aminoacid residues are improperly located on the outside of the foldedprotein. Protein activity, which can be evaluated using differentmethods, e.g., as described below, is another indicator of properprotein conformation. “Soluble, active, or both” as used herein, refersto protein that is determined to be soluble, active, or both soluble andactive, by methods known to those of skill in the art.

Interferon Activity Assays

Assays for evaluating interferon protein activity are known in the artand include binding activity assays that measure binding of interferonto a standard ligand, e.g., a Blue sepharose column or a specificantibody column.

Biological activity of interferons can be quantitated using knownassays, many of which are available commercially in kits. Most or allinterferon species have been shown to exert a similar spectrum of invitro biological activities in responsive cells, despite the existenceof different receptors for type I and type II IFN. Biological activitiesinduced by IFN include antiviral activity, which is mediated via cellreceptors and is dependent on the activation of signaling pathways, theexpression of specific gene products, and the development of antiviralmechanisms. Sensitivity of cells to IFN-mediated antiviral activity isvariable, and depends on a number of factors including cell type,expression of IFN receptors and downstream effector response elements,effectiveness of antiviral mechanisms, and the type of virus used toinfect cells.

Biological activity assays include, e.g., cytopathic effect assays(CPE), antiviral activity assays, assays that measure inhibition of cellproliferation, regulation of functional cellular activities, regulationof cellular differentiation and immunomodulation. Reporter gene assaysinclude the luciferase reporter cell-based assay described herein in theExamples. In a reporter gene assay, the promoter region of an IFNresponsive gene is linked with a heterologous reporter gene, forexample, firefly luciferase or alkaline phosphatase, and transfectedinto an IFN-sensitive cell line. Stably transfected cell lines exposedto IFN increase expression of the reporter gene product in directrelation to the dose of IFN, the readout being a measure of thisproduct's enzymic action. Many activity assay tools and kits areavailable commercially. Biological assays for interferons are described,e.g., by Meager A, “Biological assays for interferons,” 1 Mar. 2002, J.Immunol. Methods 261(1-2):21-36, incorporated herein by reference.

In embodiments, activity is represented by the % active protein in theextract supernatant as compared with the total amount assayed. This isbased on the amount of protein determined to be active by the assayrelative to the total amount of protein used in assay. In otherembodiments, activity is represented by the % activity level of theprotein compared to a standard, e.g., native protein. This is based onthe amount of active protein in supernatant extract sample relative tothe amount of active protein in a standard sample (where the same amountof protein from each sample is used in assay).

In embodiments, about 40% to about 100% of the recombinant interferonprotein is determined to be active. In embodiments, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or about 100% of therecombinant interferon protein is determined to be active. Inembodiments, about 40% to about 50%, about 50% to about 60%, about 60%to about 70%, about 70% to about 80%, about 80% to about 90%, about 90%to about 100%, about 50% to about 100%, about 60% to about 100%, about70% to about 100%, about 80% to about 100%, about 40% to about 90%,about 40% to about 95%, about 50% to about 90%, about 50% to about 95%,about 50% to about 100%, about 60% to about 90%, about 60% to about 95%,about 60% to about 100%, about 70% to about 90%, about 70% to about 95%,about 70% to about 100%, or about 70% to about 100% of the recombinantinterferon protein is determined to be active.

In other embodiments, about 75% to about 100% of the recombinantinterferon protein is determined to be active. In embodiments, about 75%to about 80%, about 75% to about 85%, about 75% to about 90%, about 75%to about 95%, about 80% to about 85%, about 80% to about 90%, about 80%to about 95%, about 80% to about 100%, about 85% to about 90%, about 85%to about 95%, about 85% to about 100%, about 90% to about 95%, about 90%to about 100%, or about 95% to about 100% of the recombinant interferonprotein is determined to be active.

Expression Systems

Methods for expressing heterologous proteins, including usefulregulatory sequences (e.g., promoters, secretion leaders, and ribosomebinding sites), in Pseudomonas host cells, as well as host cells usefulin the methods of the present invention, are described, e.g., in U.S.Pat. App. Pub. No. 2008/0269070 and U.S. patent application Ser. No.12/610,207, both titled “Method for Rapidly Screening Microbial Hosts toIdentify Certain Strains with Improved Yield and/or Quality in theExpression of Heterologous Proteins,” U.S. Pat. App. Pub. No.2006/0040352, “Expression of Mammalian Proteins in PseudomonasFluorescens,” and U.S. Pat. App. Pub. No. 2006/0110747, “Process forImproved Protein Expression by Strain Engineering,” all incorporatedherein by reference in their entirety. These publications also describebacterial host strains useful in practicing the methods of theinvention, that have been engineered to overexpress folding modulatorsor wherein protease mutations have been introduced, in order to increaseheterologous protein expression. Sequence leaders are described indetail in U.S. Patent App. Pub. No. 2008/0193974, “Bacterial leadersequences for increased expression,” and U.S. Pat. App. Pub. No.2006/0008877, “Expression systems with Sec-secretion,” both incorporatedherein by reference in their entirety, as well as in U.S. patentapplication Ser. No. 12/610,207.

Promoters

The promoters used in accordance with the present invention may beconstitutive promoters or regulated promoters. Common examples of usefulregulated promoters include those of the family derived from the lacpromoter (i.e. the lacZ promoter), especially the tac and trc promotersdescribed in U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16,Ptac17, PtacII, PlacUV5, and the T7lac promoter. In one embodiment, thepromoter is not derived from the host cell organism. In certainembodiments, the promoter is derived from an E. coli organism.

Inducible promoter sequences can be used to regulate expression ofinterferons in accordance with the methods of the invention. Inembodiments, inducible promoters useful in the methods of the presentinvention include those of the family derived from the lac promoter(i.e. the lacZ promoter), especially the tac and trc promoters describedin U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16, Ptac17, PtacII,PlacUV5, and the T7lac promoter. In one embodiment, the promoter is notderived from the host cell organism. In certain embodiments, thepromoter is derived from an E. coli organism.

Common examples of non-lac-type promoters useful in expression systemsaccording to the present invention include, e.g., those listed in Table5.

TABLE 5 Examples of non-lac Promoters Promoter Inducer P_(R) Hightemperature P_(L) High temperature Pm Alkyl- or halo-benzoates Pu Alkyl-or halo-toluenes Psal Salicylates

See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) Manual of IndustrialMicrobiology and Biotechnology (A. Demain & J. Davies, eds.) pp. 460-74(ASM Press, Washington, D.C.); H. Schweizer (2001) Current Opinion inBiotechnology, 12:439-445; and R. Slater & R. Williams (2000 MolecularBiology and Biotechnology (J. Walker & R. Rapley, eds.) pp. 125-54 (TheRoyal Society of Chemistry, Cambridge, UK)). A promoter having thenucleotide sequence of a promoter native to the selected bacterial hostcell also may be used to control expression of the transgene encodingthe target polypeptide, e.g, a Pseudomonas anthranilate or benzoateoperon promoter (Pant, Pben). Tandem promoters may also be used in whichmore than one promoter is covalently attached to another, whether thesame or different in sequence, e.g., a Pant-Pben tandem promoter(interpromoter hybrid) or a Plac-Plac tandem promoter, or whetherderived from the same or different organisms.

Regulated promoters utilize promoter regulatory proteins in order tocontrol transcription of the gene of which the promoter is a part. Wherea regulated promoter is used herein, a corresponding promoter regulatoryprotein will also be part of an expression system according to thepresent invention. Examples of promoter regulatory proteins include:activator proteins, e.g., E. coli catabolite activator protein, MalTprotein; AraC family transcriptional activators; repressor proteins,e.g., E. coli Lad proteins; and dual-function regulatory proteins, e.g.,E. coli NagC protein. Manyregulated-promoter/promoter-regulatory-protein pairs are known in theart. In one embodiment, the expression construct for the targetprotein(s) and the heterologous protein of interest are under thecontrol of the same regulatory element.

Promoter regulatory proteins interact with an effector compound, i.e., acompound that reversibly or irreversibly associates with the regulatoryprotein so as to enable the protein to either release or bind to atleast one DNA transcription regulatory region of the gene that is underthe control of the promoter, thereby permitting or blocking the actionof a transcriptase enzyme in initiating transcription of the gene.Effector compounds are classified as either inducers or co-repressors,and these compounds include native effector compounds and gratuitousinducer compounds. Manyregulated-promoter/promoter-regulatory-protein/effector-compound triosare known in the art. Although an effector compound can be usedthroughout the cell culture or fermentation, in a preferred embodimentin which a regulated promoter is used, after growth of a desiredquantity or density of host cell biomass, an appropriate effectorcompound is added to the culture to directly or indirectly result inexpression of the desired gene(s) encoding the protein or polypeptide ofinterest.

In embodiments wherein a lac family promoter is utilized, a lad gene canalso be present in the system. The lad gene, which is normally aconstitutively expressed gene, encodes the Lac repressor protein Ladprotein, which binds to the lac operator of lac family promoters. Thus,where a lac family promoter is utilized, the lad gene can also beincluded and expressed in the expression system.

Promoter systems useful in Pseudomonas are described in the literature,e.g., in U.S. Pat. App. Pub. No. 2008/0269070, also referenced above.

Other Regulatory Elements

In embodiments, soluble proteins are present in either the cytoplasm orperiplasm of the cell during production. Secretion leaders useful fortargeting proteins are described elsewhere herein, and in U.S. Pat. App.Pub. No. 2008/0193974, U.S. Pat. App. Pub. No. 2006/0008877, and in U.S.patent application Ser. No. 12/610,207, referenced above.

An expression construct useful in practicing the methods of the presentinvention can include, in addition to the protein coding sequence, thefollowing regulatory elements operably linked thereto: a promoter, aribosome binding site (RBS), a transcription terminator, andtranslational start and stop signals. Useful RBSs can be obtained fromany of the species useful as host cells in expression systems accordingto, e.g., U.S. Pat. App. Pub. No. 2008/0269070 and U.S. patentapplication Ser. No. 12/610,207. Many specific and a variety ofconsensus RBSs are known, e.g., those described in and referenced by D.Frishman et al., Gene 234(2):257-65 (8 Jul. 1999); and B. E. Suzek etal., Bioinformatics 17(12):1123-30 (December 2001). In addition, eithernative or synthetic RBSs may be used, e.g., those described in: EP0207459 (synthetic RBSs); O. Ikehata et al., Eur. J. Biochem.181(3):563-70 (1989) (native RBS sequence of AAGGAAG). Further examplesof methods, vectors, and translation and transcription elements, andother elements useful in the present invention are described in, e.g.:U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroyet al.; U.S. Pat. No. 5,281,532 to Rammler et al.; U.S. Pat. Nos.4,695,455 and 4,861,595 to Barnes et al.; U.S. Pat. No. 4,755,465 toGray et al.; and U.S. Pat. No. 5,169,760 to Wilcox.

Host Strains

Bacterial hosts, including Pseudomonas, and closely related bacterialorganisms are contemplated for use in practicing the methods of theinvention. In certain embodiments, the Pseudomonas host cell isPseudomonas fluorescens. The host cell can also be an E. coli cell.

Pseudomonas and closely related bacteria are generally part of the groupdefined as “Gram(−) Proteobacteria Subgroup 1” or “Gram-Negative AerobicRods and Cocci” (Buchanan and Gibbons (eds.) (1974) Bergey's Manual ofDeterminative Bacteriology, pp. 217-289). Pseudomonas host strains aredescribed in the literature, e.g., in U.S. Pat. App. Pub. No.2006/0040352, cited above.

For example, Pseudomonas hosts can include cells from the genusPseudomonas, Pseudomonas enalia (ATCC 14393), Pseudomonas nigrifaciensi(ATCC 19375), and Pseudomonas putrefaciens (ATCC 8071), which have beenreclassified respectively as Alteromonas haloplanktis, Alteromonasnigrifaciens, and Alteromonas putrefaciens. Similarly, e.g., Pseudomonasacidovorans (ATCC 15668) and Pseudomonas testosteroni (ATCC 11996) havesince been reclassified as Comamonas acidovorans and Comamonastestosteroni, respectively; and Pseudomonas nigrifaciens (ATCC 19375)and Pseudomonas piscicida (ATCC 15057) have been reclassifiedrespectively as Pseudoalteromonas nigrifaciens and Pseudoalteromonaspiscicida.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 16.” “Gram-negative Proteobacteria Subgroup 16” is defined asthe group of Proteobacteria of the following Pseudomonas species (withthe ATCC or other deposit numbers of exemplary strain(s) shown inparenthesis): Pseudomonas abietaniphila (ATCC 700689); Pseudomonasaeruginosa (ATCC 10145); Pseudomonas alcaligenes (ATCC 14909);Pseudomonas anguilliseptica (ATCC 33660); Pseudomonas citronellolis(ATCC 13674); Pseudomonas flavescens (ATCC 51555); Pseudomonas mendocina(ATCC 25411); Pseudomonas nitroreducens (ATCC 33634); Pseudomonasoleovorans (ATCC 8062); Pseudomonas pseudoalcaligenes (ATCC 17440);Pseudomonas resinovorans (ATCC 14235); Pseudomonas straminea (ATCC33636); Pseudomonas agarici (ATCC 25941); Pseudomonas alcaliphila;Pseudomonas alginovora; Pseudomonas andersonii; Pseudomonas asplenii(ATCC 23835); Pseudomonas azelaica (ATCC 27162); Pseudomonas beyerinckii(ATCC 19372); Pseudomonas borealis; Pseudomonas boreopolis (ATCC 33662);Pseudomonas brassicacearum; Pseudomonas butanovora (ATCC 43655);Pseudomonas cellulosa (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663);Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, ATCC 17418, ATCC17461); Pseudomonas fragi (ATCC 4973); Pseudomonas lundensis (ATCC49968); Pseudomonas taetrolens (ATCC 4683); Pseudomonas cissicola (ATCC33616); Pseudomonas coronafaciens; Pseudomonas diterpeniphila;Pseudomonas elongata (ATCC 10144); Pseudomonasflectens (ATCC 12775);Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella;Pseudomonas corrugata (ATCC 29736); Pseudomonas extremorientalis;Pseudomonas fluorescens (ATCC 35858); Pseudomonas gessardii; Pseudomonaslibanensis; Pseudomonas mandelii (ATCC 700871); Pseudomonas marginalis(ATCC 10844); Pseudomonas migulae; Pseudomonas mucidolens (ATCC 4685);Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha(ATCC 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas veronii(ATCC 700474); Pseudomonas frederiksbergensis; Pseudomonas geniculata(ATCC 19374); Pseudomonas gingeri; Pseudomonas graminis; Pseudomonasgrimontii; Pseudomonas halodenitrificans; Pseudomonas halophila;Pseudomonas hibiscicola (ATCC 19867); Pseudomonas huttiensis (ATCC14670); Pseudomonas hydrogenovora; Pseudomonas jessenii (ATCC 700870);Pseudomonas kilonensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonaslini; Pseudomonas marginata (ATCC 25417); Pseudomonas mephitica (ATCC33665); Pseudomonas denitrificans (ATCC 19244); Pseudomonaspertucinogena (ATCC 190); Pseudomonas pictorum (ATCC 23328); Pseudomonaspsychrophila; Pseudomonas filva (ATCC 31418); Pseudomonas monteilii(ATCC 700476); Pseudomonas mosselii; Pseudomonas oryzihabitans (ATCC43272); Pseudomonas plecoglossicida (ATCC 700383); Pseudomonas putida(ATCC 12633); Pseudomonas reactans; Pseudomonas spinosa (ATCC 14606);Pseudomonas balearica; Pseudomonas luteola (ATCC 43273); Pseudomonasstutzeri (ATCC 17588); Pseudomonas amygdali (ATCC 33614); Pseudomonasavellanae (ATCC 700331); Pseudomonas caricapapayae (ATCC 33615);Pseudomonas cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC35104); Pseudomonas fuscovaginae; Pseudomonas meliae (ATCC 33050);Pseudomonas syringae (ATCC 19310); Pseudomonas viridiflava (ATCC 13223);Pseudomonas thermocarboxydovorans (ATCC 35961); Pseudomonasthermotolerans; Pseudomonas thivervalensis; Pseudomonas vancouverensis(ATCC 700688); Pseudomonas wisconsinensis; and Pseudomonas xiamenensis.

The host cell can also be selected from “Gram-negative ProteobacteriaSubgroup 17.” “Gram-negative Proteobacteria Subgroup 17” is defined asthe group of Proteobacteria known in the art as the “fluorescentPseudomonads” including those belonging, e.g., to the followingPseudomonas species: Pseudomonas azotoformans; Pseudomonas brenneri;Pseudomonas cedrella; Pseudomonas corrugata; Pseudomonasextremorientalis; Pseudomonas fluorescens; Pseudomonas gessardii;Pseudomonas libanensis; Pseudomonas mandelii; Pseudomonas marginalis;Pseudomonas migulae; Pseudomonas mucidolens; Pseudomonas orientalis;Pseudomonas rhodesiae; Pseudomonas synxantha; Pseudomonas tolaasii; andPseudomonas veronii.

Codon Optimization

Methods for optimizing codons to improve expression in bacterial hostsare known in the art and described in the literature. For example,optimization of codons for expression in a Pseudomonas host strain isdescribed, e.g., in U.S. Pat. App. Pub. No. 2007/0292918, “CodonOptimization Method,” incorporated herein by reference in its entirety.

Codon optimization for expression in E. coli is described, e.g., byWelch, et al., 2009, PLoS One, “Design Parameters to Control SyntheticGene Expression in Escherichia coli, 4(9): e7002, Ghane, et al., 2008,“Overexpression of Biologically Active Interferon B Using Synthetic Genein E. coli,” Journal of Sciences, Islamic Republic of Iran 19(3):203-209, and Valente, et al., 2004, “Translational Features of HumanAlpha 2b Interferon Production in Escherichia coli,” Applied andEnvironmental Microbiology 70(8): 5033-5036, all incorporated byreference herein.

Fermentation Format

The expression system according to the present invention can be culturedin any fermentation format. For example, batch, fed-batch,semi-continuous, and continuous fermentation modes may be employedherein.

In embodiments, the fermentation medium may be selected from among richmedia, minimal media, and mineral salts media. In other embodimentseither a minimal medium or a mineral salts medium is selected. Incertain embodiments, a mineral salts medium is selected.

Mineral salts media consists of mineral salts and a carbon source suchas, e.g., glucose, sucrose, or glycerol. Examples of mineral salts mediainclude, e.g., M9 medium, Pseudomonas medium (ATCC 179), and Davis andMingioli medium (see, B D Davis & E S Mingioli (1950) J. Bact.60:17-28). The mineral salts used to make mineral salts media includethose selected from among, e.g., potassium phosphates, ammonium sulfateor chloride, magnesium sulfate or chloride, and trace minerals such ascalcium chloride, borate, and sulfates of iron, copper, manganese, andzinc. Typically, no organic nitrogen source, such as peptone, tryptone,amino acids, or a yeast extract, is included in a mineral salts medium.Instead, an inorganic nitrogen source is used and this may be selectedfrom among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia.A mineral salts medium will typically contain glucose or glycerol as thecarbon source. In comparison to mineral salts media, minimal media canalso contain mineral salts and a carbon source, but can be supplementedwith, e.g., low levels of amino acids, vitamins, peptones, or otheringredients, though these are added at very minimal levels. Media can beprepared using the methods described in the art, e.g., in U.S. Pat. App.Pub. No. 2006/0040352, referenced and incorporated by reference above.Details of cultivation procedures and mineral salts media useful in themethods of the present invention are described by Riesenberg, D et al.,1991, “High cell density cultivation of Escherichia coli at controlledspecific growth rate,” J. Biotechnol. 20 (1):17-27.

Fermentation may be performed at any scale. The expression systemsaccording to the present invention are useful for recombinant proteinexpression at any scale. Thus, e.g., microliter-scale, centiliter scale,and deciliter scale fermentation volumes may be used, and 1 Liter scaleand larger fermentation volumes can be used.

In embodiments, the fermentation volume is at or above about 1 Liter. Inembodiments, the fermentation volume is about 1 liter to about 100liters. In embodiments, the fermentation volume is about 1 liter, about2 liters, about 3 liters, about 4 liters, about 5 liters, about 6liters, about 7 liters, about 8 liters, about 9 liters, or about 10liters. In embodiments, the fermentation volume is about 1 liter toabout 5 liters, about 1 liter to about 10 liters, about 1 liter to about25 liters, about 1 liter to about 50 liters, about 1 liter to about 75liters, about 10 liters to about 25 liters, about 25 liters to about 50liters, or about 50 liters to about 100 liters In other embodiments, thefermentation volume is at or above 5 Liters, 10 Liters, 15 Liters, 20Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or50,000 Liters.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1 Production of rIFN-β from High Throughput ExpressionSamples

In the following experiment, IFN-β C17S expression strains wereconstructed, and the amount of IFN-β in the insoluble fraction obtainedfor each was quantitated. Based on the resulting data, certain strainswere selected for use in optimizing the non-denaturing extractionprocess of the present invention.

Construction and Growth of IFN-β Expression Strains

The IFN-β 1b coding sequence was constructed using P. fluorescenspreferred codons to encode for the human IFN-β amino acid sequencecorresponding to the therapeutic Betaseron. FIG. 7 shows the amino acidand DNA sequences of the synthetic IFN-β (Betaseron) gene.

Plasmids were constructed which carry the codon-optimized IFN-β fused tonineteen P. fluorescens secretion leaders. The secretion leaders wereincluded to target the protein to the periplasm where it may berecovered in the properly folded and active form. In addition, oneplasmid was constructed which carries the codon-optimized IFN-β designedfor cytoplasmic expression.

Expression of IFN-β was driven from the Ptac promoter and translationinitiated from either a high (Hi) or medium (Med) activity ribosomebinding site (RBS). The resulting 20 plasmids were transformed into 30P. fluorescens host strains (16 protease deletion strains, 13 foldingmodulator overexpression strains and 1 wild type strain) to produce 600expression strains (see Tables 6 and 7). Folding modulators, whenpresent, were encoded on a second plasmid and expression driven by amannitol inducible promoter.

The thirty host strains carrying each of 20 IFN-β expression plasmids(600 expression hosts in total) were grown in triplicate in 96-wellplates. Samples harvested 24 hours after induction were used foranalysis.

Expression of IFN-β Using Pfēnex Expression Technology in 96-Well Format

Each plasmid (Table 6) was transformed into 30 P. fluorescens hoststrains (Table 7) as follows: Twenty-five microliters of competent cellswere thawed and transferred into a 96-well electroporation plate (BTXECM630 Electroporator), and 1 microliter miniprep plasmid DNA was addedto each well. Cells were electroporated at 2.5 KV, 200 Ohms, and 25 μF.Cells were resuspended in 75 microliters HTP-YE media with traceminerals, transferred to 96-well deep well plate with 500 μl M9 salts 1%glucose medium (seed culture), and incubated at 30° C., shaking 300 rpmand 50-mm diameter throw for 48 hours.

Ten microliters of seed culture were transferred into triplicate wellsof 96-well deep well plates, each well containing 500 microliters ofHTP-YE medium, and incubated as before for 24 hours.Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to each well fora final concentration of 0.3 mM to induce the expression of IFN-β. Forgrowth of small cultures in HTP microwells, a specific culture pH is nottightly controlled and the cell density can differ slightly from well towell. Mannitol (Sigma, M1902) was added to each well at a finalconcentration of 1% to induce the expression of folding modulators infolding modulator over-expressing strains, and the temperature wasreduced to 25° C. Twenty four hours after induction, cultures werecollected for analysis. For OD normalization, cells were mixed withsterile 1×PBS to obtain a final OD600=20 in a final volume of 400microliters using the Biomek liquid handling station (Beckman Coulter).Samples were collected in cluster tube racks.

Sample Preparation and SDS-CGE Analysis

Soluble fractions (supernatants obtained after centrifugation oflysates) and insoluble fractions (pellets obtained after centrifugationof lysates) were prepared by sonicating the OD-normalized cultures,followed by centrifugation. Frozen, normalized culture broth (400microliters) was thawed and sonicated for 3.5 minutes. The lysates werecentrifuged at 20,800×g for 20 minutes (4° C.) and the soluble fractionsremoved using manual or automated liquid handling. The insolublefractions were frozen and then thawed for re-centrifugation at 20,080×gfor 20 minutes at 4° C., to remove residual supernatant. The insolublefractions were then resuspended in 400 μL of 1× phosphate bufferedsaline (PBS), pH 7.4. Further dilutions of soluble and insolublefractions for SDS-CGE analysis were performed in 1× phosphate bufferedsaline (PBS), pH 7.4. Soluble and insoluble fractions were prepared foranalysis by SDS capillary gel electrophoresis (CGE) (Caliper LifeSciences, Protein Express LabChip Kit, Part 760301), in the presence ofdithiothreitol (DTT).

Normalized soluble and insoluble fractions from each well of the 600strains expressing IFN-β were analyzed by reducing SDS-CGE analysis inone replicate for the soluble fractions and insoluble fractions. NoIFN-β signal was detected in the soluble fractions. IFN-β signal variedfrom no signal to greater than 400 mg/L in the insoluble fractions. Onlyfive of the twenty plasmids tested showed measurable signal of IFN-β inthe insoluble fractions of all thirty host strains: p530-001, p530-007,p530-011, p530-018 and p530-020. Valley to valley integration of IFN-βsignal using Caliper LabChipGX software was performed in all 150 strainsconsisting of the five plasmids listed above in the thirty host strains,and data were used to calculate volumetric yields. Volumetric yields ofthe 150 strains analyzed ranged from 2 to 482 mg/L. Strains carryingp530-020 attained consistently higher yields of IFN-β in the insolublefraction than other expression strains; however, the protein migratedhigher than expected on SDS-CGE, indicating that the secretion leaderwas not cleaved. High yields were also observed with 2 host strainscarrying p530-001. No significant difference in IFN-β in the insolublefraction was observed among the 30 strains except potentially in onestrain, DC441, a Ion hslUV protease deletion strain, which showedsomewhat higher yields than the other 29 strains.

A subset of 17 top expression strains (Table 8), excluding strainscontaining plasmid p530-020, was selected for further analyses. Theexpression strains containing plasmid p530-020 were excluded fromfurther consideration in this experiment due to the potentiallyunprocessed leader. SDS-CGE analysis was performed on the soluble andinsoluble fractions for these strains. Quantification of the SDS-CGEoutput is shown in Table8. IFN-β protein concentration ranged from 102to greater than 482 mg/L. Based upon insoluble yield and processing ofeither the periplasmic leader sequence or the N-terminal Met from IFN-β,strains were chosen to proceed to fermentation assessment.

TABLE 6 Plasmids Expression Secretion Plasmid Vector Leader RBS p530-001pDOW5271 None Hi p530-002 pDOW5204 Pbp Med p530-003 pDOW5206 DsbA Hip530-004 pDOW5207 DsbA Med p530-005 pDOW5209 Azu Hi p530-006 pDOW5210Azu Med p530-007 pDOW5217 LAO Hi p530-008 pDOW5220 Ibp-S31A Hi p530-009pDOW5223 TolB Hi p530-010 pDOW5226 Trc Hi p530-011 pDOW5232 Ttg2C Hip530-012 pDOW5235 FlgI Hi p530-013 pDOW5238 CupC2 Hi p530-014 pDOW5241CupB2 Hi p530-015 pDOW5244 CupA2 Hi p530-016 pDOW5247 NikA Hi p530-017pDOW5256 PorE Hi p530-018 pDOW5259 Pbp-A20V Hi p530-019 pDOW5262 DsbC Hip530-020 pDOW5265 Bce Hi

TABLE 7 IFN-β Expression Strains Strain Strain Name Description DC454Wild type DC441 PD DC462 FMO DC468 PD DC469 PD DC485 PD DC486 PD DC487PD DC488 PD DC489 PD DC490 PD DC491 PD DC492 PD DC498 PD DC538 FMO DC539FMO DC544 FMO DC547 FMO DC548 FMO DC552 FMO DC565 FMO DC566 FMO DC567FMO DC568 FMO DC575 FMO DC584 FMO DC598 FMO DC599 FMO DC667 FMO DC954 PDPD = protease deletion strain, FMO = folding modulator over-expressionstrain

TABLE 8 Calculated Volumetric IFN-β Yields of Top 17 Strains by SDS-CGEStrain Name Vol. Yield >100 ug/ml Plasmid Host Strain Leader PS530-001482.3 p530-001 DC441 x PS530-101 216.5 p530-001 DC485 x PS530-011 161.1p530-011 DC441 ttg2C PS530-071 148.8 p530-011 DC468 ttg2C PS530-007141.2 p530-007 DC441 Lao PS530-031 131.3 p530-011 DC454 ttg2C PS530-201122.6 p530-001 DC490 x PS530-531 121.0 p530-011 DC598 ttg2C PS530-211119.8 p530-011 DC490 ttg2C PS530-151 119.8 p530-011 DC487 ttg2CPS530-061 119.6 p530-001 DC468 x PS530-411 114.0 p530-011 DC565 ttg2CPS530-231 113.3 p530-011 DC491 ttg2C PS530-391 112.2 p530-011 DC552ttg2C PS530-027 104.5 p530-007 DC454 Lao PS530-291 103.3 p530-011 DC538ttg2C PS530-271 102.2 p530-011 DC498 ttg2C

Example 2 Extraction of IFN-β 1b from High Throughput ExpressionMaterial

IFN-β 1b was successfully extracted from insoluble fractions from HTPexpression cultures, using extraction conditions comprising Zwittergent3-14 detergent.

HTP expression plate cultures of Pseudomonas fluorescens strainsPS530-001 overexpressing cytoplasmic IFN-β 1b and 530-220,overexpressing secreted IFN-β 1b (described in Example 1), weresonicated and centrifuged to obtain an insoluble fraction and a solublefraction. The pellets were resuspended in extraction buffer 1×PBS, pH7.4 or sodium acetate at pH 4.5. Each buffer was tested either with orwithout Zwittergent 3-14 detergent at 1% (w/v). Each of the fourcombinations of buffer and detergent was incubated for 1-2 hours at roomtemperature or overnight at 4° C. with shaking. After extraction, eachsample was centrifuged for 20 minutes at 20,080×g at 4° C. to produce asecond insoluble pellet fraction (extract pellet) and a second solublesupernatant fraction (extract supernatant). The first insoluble fractionand first soluble fraction, and the extract pellet fraction and extractsupernatant fraction, were analyzed by SDS-CGE. The results are shown inFIGS. 1A and 1B. As seen in Lane 7, the extraction condition includingPBS buffer and Zwittergent 3-14 yielded soluble IFN-|3.

Example 3 Optimization of Conditions for Extraction

Insoluble fractions from fermentation cultures were extracted underconditions comprising different detergents.

Frozen cell paste from a 1 L fermentation (grown at 32° C., pH 6.5, andinduced using 0.2 mM IPTG at an OD₅₇₅ of 100) of strain PS530-001,overexpressing recombinant IFN-β 1b, was resuspended in lysis buffercontaining 20 mM sodium phosphate (JT Baker), pH 7.4 to a final solidsconcentration of 20% (w/v). The well-mixed cell slurry was lysed withtwo passes at 38 kpsi through a Constant cell disruptor (ConstantSystems, Inc.). The lysate was split in half, and spun by centrifugationat 15,000×g for 30 minutes at 4° C. (Beckman Coulter, PN# J-20, XPF).The pellets (containing IFN-β and cell debris) were resuspended and eachwas washed in either Buffer A (20 mM sodium phosphate, pH 7.4) or BufferB (20 mM sodium acetate, pH 4.0). Samples were spun by centrifugationunder the same conditions described for the first spin, supernatantswere removed, and the pellets were again resuspended in either Buffer Aor B at 20% solid concentration. For each buffer, twenty aliquots of 1mL each were placed in 1.5 mL conical tubes. Detergent stock solutionswere added to the conical tubes at different concentrations. All tubeswere incubated at room temperature for 1 hour or overnight (18 hours) at4° C. with continuous mixing. After extraction, the solutions werecentrifuged and the extract supernatant fractions were analyzed forprotein concentration by SDS-CGE. FIG. 2 provides a flow chart showinghow the sample preparation and extraction were carried out.

Of the detergents tested, Zwittergent 3-14 and N-lauroylsarcosine (NLS),were found to give the best yields regardless of buffer and incubationtime (Table 9). However, the product extracted using NLS was not active,as determined by its inability to bind to either a Blue Sepharoseaffinity column or a cation exchange column (SP HP) (data not shown).The product extracted using Zwittergent 3-14 was determined to beactive.

TABLE 9 Evaluation of Detergents for Extraction Detergent ExtractedProduct Concentration (ug/mL) Concentration Buffer A Buffer B Detergent(w/v) 1 hr @RT 18 hr @RT 1 hr @RT 18 hr @RT Zwittergent 3-14 0.50% 748557 1011 734 1.00% 731 392 1060 936 2.00% 903 398 1548 1146Lauroylsarcosine 0.20% 1023 643 NA NA 0.50% 3104 2125 324 150 1.50% 27822670 2319 2668 NDSB195 10.00% 8 6 11 46 15.00% 14 13 31 119 NDSB2565.00% 20 56 15 43 15.00% 204 233 114 135 Chaps 0.50% 11 36 98 160 2.00%75 170 179 250 Octylglucopyranoside 1.00% 83 175 121 169 5.00% 196 258164 215 Sodium 0.50% 129 237 NA NA Deoxycholate 1.00% 196 274 NA NATween-20 0.05% 4 11 NA 6 0.50% 11 37 3 18 Tween-80 0.01% 4 6 NA 7 0.10%5 10 NA 12 0.50% 7 25 3 21 Triton-100 0.10% 25 68 33 103 1.00% 40 85 62176

Evaluation of Zwittergent Analogs

Using similar methods, Zwittergent analogs were evaluated for theirextraction efficiency. The results are shown in Table 10. The bestyields were observed with Zwittergent 3-14. Zwittergent 3-12,Zwittergent 3-10, and Zwittergent 3-08 were also effective.

TABLE 10 Evaluation of Zwittergent Analogs for Extraction of IFN-β 1bDetergent Detergent Conc. Solid Conc. Protein (ug/mL) Zwittergent 3-08 10% 20% 292 Zwittergent 3-10 1.0% 20% 233 Zwittergent 3-12 1.0% 20% 357Zwittergent 3-16 0.1% 20% 17 Zwittergent 3-14 1.0% 20% 430 Zwittergent3-14 1.0% 10% 396 Zwittergent 3-14 1.0%  5% 548

Evaluation of the Zwittergent 3-14 Concentration

To efficiently solubilize proteins, the detergent concentrationtypically needs to be above its CMC value. The CMC of Zwittergent 3-14is about 0.01% w/v. Extraction conditions including sodium phosphatebuffer at pH 7.4 with increasing concentrations of Zwittergent 3-14 wereevaluated. The cell paste used was obtained by growing strain PS530-001at 32° C., pH 6.5, and induced using 0.2 mM IPTG at an OD₅₇₅ of 100. Theresults in Table 11 show that use of Zwittergent 3-14 at 1% (w/v)concentration resulted in the highest extraction yield.

TABLE 11 Effect of Zwittergent 3-14 Concentration on Extraction of IFN-β1b Extraction Yield Zwittergent 3-14 % IFN-β protein extracted fromConcentration Extraction Yield insoluble pellet (% w/v) microgram/mL(insoluble fraction) 0.01% 10 0% 0.05% 36 1% 0.10% 72 2% 0.50% 341 9%1.00% 787 21%  2.00% 620 17% 

Evaluation of Additional Chemical Reagents

As shown in Table 11, extraction conditions including Zwittergent 3-14at 1% (w/v) concentration in sodium phosphate buffer at pH 7.4 yielded21% of the IFN-β 1b detected in the original insoluble fraction. Furtheroptimization was conducted.

High concentration (e.g., 6 to 8 M) of some chaotropic reagents likeurea and guanidinium hydrochloride commonly have been used as a strongdenaturant for solubilization of inclusion bodies. Chaotropes such asurea can increase the detergent critical micelle concentration (CMC) andmay potentially improve the extraction efficiency. Low concentrations ofurea (up to 2 M) were evaluated in the extraction conditions. Salts,e.g., NaCl, can also affect detergent CMC. Varying Zwittergent 3-14concentrations were evaluated due to the potential interplay betweendetergent CMC and the presence of chaotrophic reagents and salts. Theconcentration of insoluble inclusion solids in the extraction conditionswas also varied. Lower solids concentration than the 20% (w/v)previously used were evaluated.

In summary, the effect of varying the following parameters on extractionefficiency was tested.

Sodium Chloride: 150-1850 mM

Urea: 0-2 M

Zwittergent 3-14: 0.1-1.0% w/v

Solids: 5-20% w/v

pH: 6.5-8.5

The flow chart in FIG. 3 describes the preparation and extraction of thefirst insoluble pellet fraction for this optimization study. Table 12shows the result of the study. FIGS. 4A and B summarize the results andsignificance of the effect of each parameter on the extraction yield.For optimization of extraction of interferon 0 from the insolublefraction, a two-level five-factor half-fractional factorial experimentaldesign was used. JMP software (SAS Institute, Cary, N.C.) was used forexperimental design and analysis. The software estimates the effect ofindividual factors as well as interactions on experimental output(amount of interferon extracted).

TABLE 12 Results of Extraction Study Interferon-β in extract Solids NaClUrea Z314 supernatant No. (%) pH (M) (M) (%) (mg/L) 1 −−−−+ 5 6.5 0.15 01 2275 2 −−−+− 5 6.5 0.15 2 0.1 896 3 −−+−− 5 6.5 1.85 0 0.1 246 4 −−+++5 6.5 1.85 2 1 7024 5 −+−−− 5 8.5 0.15 0 0.1 638 6 −+−++ 5 8.5 0.15 2 15614 7 −++−+ 5 8.5 1.85 0 1 5414 8 −+++− 5 8.5 1.85 2 0.1 1711 9 0 12.57.5 1 1 0.55 3362 10 0 12.5 7.5 1 1 0.55 3693 11 0 12.5 7.5 1 1 0.553809 12 +−−−− 20 6.5 0.15 0 0.1 65 13 +−−++ 20 6.5 0.15 2 1 2345 14+−+−+ 20 6.5 1.85 0 1 2149 15 +−++− 20 6.5 1.85 2 0.1 438 16 ++−−+ 208.5 0.15 0 1 2350 17 ++−+− 20 8.5 0.15 2 0.1 677 18 +++−− 20 8.5 1.85 00.1 199 19 +++++ 20 8.5 1.85 2 1 4486

Based on the above data, an optimized extraction condition was selectedfor experiments described hereinafter: 1% (w/v) Zwittergent 3-14, 2 MUrea, 2 M NaCl, Solids 5% w/v, buffer pH 7.5 to 8.5. Using theseoptimized conditions, the observed extraction yield (in the extractsupernatant) was found to be consistently 90% or above (i.e., 90% ormore of the amount of recombinant protein measured in the insolublefraction).

Example 4 Production of rIFN-β 1b from Large Scale Fermentation

Production of recombinant human-β interferon (IFN-β 1b) protein inPseudomonas fluorescens Pfēnex Expression Technology™ strain PS530-001was successfully achieved in 2 liter fermentors. Multiple fermentationconditions were evaluated resulting in expression of IFN-β 1b up to 9.2g/L.

Fermentation cultures were grown in 2 liter fermentors containing amineral salts medium (as described herein and also by, e.g., Riesenberg,D., et al., 1991). Culture conditions were maintained at 32° C. and pH6.5 through the addition of aqueous ammonia. Dissolved oxygen wasmaintained in excess through increases in agitation and flow of spargedair and oxygen into the fermentor. Glycerol was delivered to the culturethroughout the fermentation to maintain excess levels. These conditionswere maintained until the target culture optical density (A575) forinduction was reached, at which time IPTG was added to initiate IFN-βproduction. The optical density at induction, the concentration of IPTG,pH and temperature were all varied to determine optimal conditions forexpression. After 24 hours, the culture from each fermentor washarvested by centrifugation and the cell pellet frozen at −80° C.

Fermentation cultures were induced at 100 OD₅₇₅ using 0.2 mM IPTG, at pH6.5 and a temperature of 32° C. Replicate fermentations resulted inIFN-β production at 7.5, 8.4 and 7.9 g/L as determined by SDS-CGE of theinitial insoluble fraction (FIG. 5). When these insoluble fractions weresubjected to extraction (under conditions including 1% (w/v) Zwittergent3-14, 2 M Urea, 2 M NaCl, Solids 5% w/v, and buffer pH 8.2), solubilizedIFN-β were observed in the extract supernatant at 2.2, 2.4, and 2.6 g/L.This represents an average extraction yield of 31%.

Increasing the induction OD to 120 to 160, and decreasing thefermentation pH to 5.7 to 6.25, increased IFN-β titers in the initialinsoluble fraction to 8.8-9.2 g/L (FIG. 6). Extraction of these cellpellets (using the same extraction conditions as for the experimentshown in FIG. 5) resulted in 3.1 to 4.0 g/L of IFN-β in the extractedsupernatant fraction, an average extraction yield of 39% (Table 13).

TABLE 13 Extracted Solubilized IFN-β Based on Induction ConditionsInduction OD of 100 Induction OD of 120-160 and pH 6.5 and pH 5.7 to6.25 Total Extracted Total Extracted Insoluble Solubilized ExtractedInsoluble Solubilized Extracted Titer (g/L) Titer (g/L) Yield (%) Titer(g/L) Titer (g/L) Yield (%) u2 7.5 2.2 29 u2 9.2 4.0 43 u7 8.4 2.4 29 u38.8 3.1 35 u8 7.9 2.6 33 u5 8.8 ND ND average 7.9 2.4 31 average 8.9 3.539 std dev 0.4 0.2 2.3 std dev 0.3 0.6 5.6

Example 5 Activity Analysis of IFN-β Extraction Product

Broth from fermentation of Pseudomonas fluorescens strain PS530-001 (1 Lfermentation at 32° C., pH 6.0, induced at OD₅₇₅ of 100 using 0.2 mMIPTG) was centrifuged and the supernatant discarded. The cell paste wasresuspended in 20 mM Tris, pH 8.2 (in a ratio of 1:4) and lysed bypassing through Microfluidics Microfluidizer M110Y at 15,000 psi. Thelysate was centrifuged and the soluble fraction discarded. The insolublefraction was mixed with extraction buffer (20 mM Tris, 2 M NaCl, 2 Murea, 1% Zwittergent 3-14, pH 8.2) at room temperature for 1 hour andcentrifuged to produce an extract supernatant fraction and an extractpellet fraction. The extraction yield of IFN-β (IFN-β in extractsupernatant fraction/IFN-β in the initial insoluble fraction) was closeto 100% (>99%) based on SDS-CGE analysis (data not shown).

The extract supernatant was filtered and loaded on a 5 mL GE HealthcareBlue Sepharose column equilibrated with 20 mM Tris, 2 M NaCl, pH 8.2.The column was washed with the same buffer and the IFN-β eluted with 20mM Tris, 2 M NaCl, 50% propylene glycol, pH 8.2. The protein in theelution pool was analyzed by SDS-CGE and found to be more than 98% pureIFN-β. Aliquots of the elution pool were exchanged into Buffers C (5 mMglycine pH 3.0) and D (5 mM aspartic acid, 9% trehalose, pH 4.0).

The exchanged samples were analyzed by SDS-CGE as well as with acell-based assay (PBL Interferon Source, #51100-1). The cell-based assayuses a human cell line (PIL5) sensitized with IFN-type 1 receptor. IFN-βbinds to the receptor, which sends a signal via the Jak1/STAT1 signaltransduction pathway, activating ISG15-luciferase transcription via theInterferon Sensitive Response Element (ISRE). Cell-based assay kitinstructions were followed as per manufacturer's protocol (51100 rev01).The signal was read using conventional plate readers with luminescencedetection. Table 14 summarizes the SDS-CGE and cell-based assay results,which indicate that the IFN-β in the samples was fully active.

TABLE 14 Results of Activity Assays Sample SDS-CGE (mg/L) Cell-basedassay (mg/L) Blue-Sepharose Elution 436 477 pool in Exchange Buffer ABlue-Sepharose Elution 404 404 pool in Exchange Buffer B

Example 6 Production of IFN-α 2a and 2b from High Throughput ExpressionSamples

IFN-α 2a and IFN-α 2b coding sequences were constructed using P.fluorescens preferred codons to encode for the human proteins. FIG. 8shows the amino acid and DNA sequences of the synthetic IFN-α 2a gene,and FIG. 9 shows the amino acid and DNA sequences of the synthetic IFN-α2b gene.

Plasmids expressing either protein were constructed and transformed intodifferent host strains. Expression strains were tested for their abilityto express recombinant protein using HTP analysis, as described withregard to IFN-β herein. A subset of the expression strains are selectedfor fermentation studies.

The selected strains were grown and induced according to the presentinvention. The cells were centrifuged, lysed, and centrifuged again asdescribed herein for IFN-β. The resulting insoluble fraction and firstsoluble fraction were extracted using extraction conditions describedherein. The resulting IFN-α 2a and IFN-α 2b extract supernatants werequantitated using SDS-CGE (data not shown).

Example 7 Extraction of IFN-α 2a and 2b from High Throughput ExpressionMaterial

The first insoluble fraction obtained as described in Example 6 isextracted using the extraction conditions of the present invention.IFN-α 2a and 2b in the resulting second soluble fractions are evaluatedby CGE and bioactivity assay.

Example 8 Production of IFN-α 2a and 2b from Large Scale Fermentation

IFN-α 2a and 2b expressing strains selected by HTP analysis are grown in2 liter fermentors using optimized fermentation conditions of thepresent invention, e.g., as described in Example 4. The first insolublefraction is extracted using the methods of the present invention, e.g.,as described in Example 4. The IFN-α 2a and 2b protein present in thefirst insoluble and second soluble fractions are evaluated by CGE andcompared.

Example 9 Analysis of IFN-α 2a and 2b Extraction Product

The extraction product obtained in Example 8 is analyzed for IFN-α 2aand 2b bioactivity.

What is claimed is:
 1. A method for producing a recombinant Type 1interferon protein, said method comprising: expressing the recombinantinterferon protein by culturing a Pseudomonas or E. coli host cellcontaining an expression construct comprising a coding sequence that hasbeen optimized for expression in the host cell; lysing the host cell;obtaining an insoluble fraction and a soluble fraction from the lysisstep; extracting the insoluble fraction by subjecting it tonon-denaturing extraction conditions; and obtaining an extract pelletand an extract supernatant from the insoluble fraction; wherein therecombinant protein in the extract supernatant is present in solubleform, active form, or a combination thereof, without being furthersubjected to a renaturing or refolding step.
 2. The method of claim 1,wherein the non-denaturing extraction conditions comprise the presenceof a mild detergent.
 3. The method of claim 2, wherein the milddetergent is a Zwitterionic detergent.
 4. The method of claim 3, whereinthe Zwitterionic detergent is Zwittergent 3-08, Zwittergent 3-10,Zwittergent 3-12, or Zwittergent 3-14.
 5. The method of claim 4, whereinthe non-denaturing extraction conditions comprise about 0.5% to about 2%Zwittergent 3-14.
 6. The method of claim 2, wherein the non-denaturingextraction conditions further comprise a chaotropic agent and acosmotropic salt.
 7. The method of claim 6, wherein the chaotropic agentis urea or guanidinium hydrochloride, and wherein the cosmotropic saltis NaCl, KCl, or (NH₄)₂SO₄.
 8. The method of claim 7, wherein thenon-denaturing extraction conditions comprise: about 0.5 to about 2%Zwittergent 3-14; about 0 to about 2 M urea; about 0 to about 2 M NaCl;and wherein the pH is about 6.5 to about 8.5.
 9. The method of claim 8,wherein the non-denaturing extraction conditions comprise: about 1%Zwittergent 3-14; about 2 M urea; about 2 M NaCl; and wherein the pH isabout 8.2.
 10. The method of claim 9, wherein the non-denaturingextraction conditions additionally comprise about 5% w/v solids.
 11. Themethod of claim 8, wherein the non-denaturing extraction conditionsadditionally comprise about 1% to about 40% w/v solids.
 12. The methodof claim 1, wherein the recombinant Type 1 interferon protein is aninterferon-β, an interferon-α, an interferon-κ, or an interferon-ω. 13.The method of claim 12, wherein the recombinant Type 1 interferonprotein is an interferon-β, and wherein said interferon-β is selectedfrom the group consisting of: a human interferon-β 1b and humaninterferon-β 1b C17S.
 14. The method of claim 12, wherein therecombinant Type 1 interferon protein is an interferon-α, and whereinthe interferon-α is selected from the group consisting of: humaninterferon-α2a and human interferon-α 2b.
 15. The method of claim 1,further comprising measuring the amount of recombinant Type 1 interferonprotein in the insoluble fraction and the extract supernatant fractions,wherein the amount of recombinant interferon protein detected in theextract supernatant fraction is about 10% to about 95% of the amount ofthe recombinant interferon protein detected in the insoluble fraction.16. The method of claim 1, further comprising measuring the activity ofthe recombinant protein, wherein about 40% to about 100% of therecombinant protein present in the extract supernatant is determined tobe active when compared with the total amount of recombinant proteinassayed.
 17. The method of claim 1, wherein the recombinant protein inthe extract supernatant is present at a concentration of about 0.3 gramsper liter to about 10 grams per liter.
 18. The method of claim 1,wherein the host cell is cultured in a volume of about 1 to about 20 ormore liters.
 19. The method of claim 1, wherein the expression constructcomprises an inducible promoter.
 20. The method of claim 19, wherein theexpression construct comprises a lac promoter derivative and expressionof the interferon is induced by IPTG.
 21. The method of claim 20,wherein the host cell is grown at a temperature of about 25° C. to about33° C., at a pH of about 5.7 to about 6.5, and wherein the IPTG is addedto a final concentration of about 0.08 mM to about 0.4 mM, when theOD₅₇₅ has reached about 80 to about
 160. 22. The method of claim 21,wherein the host cell is grown at a temperature of about 32° C., at a pHof about 5.7 to 6.25, and wherein the IPTG is added to a finalconcentration of about 0.2 mM, when the OD₅₇₅ has reached about 120 toabout
 160. 23. The method of claim 1 wherein the expression constructcomprises a high activity ribosome binding site.
 24. The method of claim1 wherein the host cell is a lon hslUV protease deletion strain.
 25. Themethod of claim 13, wherein the human interferon-β 1b or humaninterferon-β 1b C17S is expressed in the cytoplasm of the host cell. 26.A method for extracting a recombinant Type 1 interferon protein, whereinthe recombinant interferon protein is present in an insoluble fraction,said insoluble fraction produced after lysis of a Pseudomonas or E. colihost cell expressing the recombinant interferon protein, said methodcomprising: subjecting the insoluble fraction to non-denaturingextraction conditions; and obtaining an extract pellet from theinsoluble fraction, said extract pellet comprising recombinantinterferon protein; wherein the recombinant interferon protein in theextract pellet is in soluble form, active form, or a combinationthereof, without being subjected to a renaturing or refolding step. 27.A method for producing an insoluble fraction comprising a recombinantType 1 interferon protein, wherein the recombinant interferon protein isexpressed in a Pseudomonas or E. coli host cell from a nucleic acidconstruct comprising a nucleic acid sequence that is operably linked toa lac derivative promoter, said method comprising: growing the host cellat a temperature of about 25° C. to about 33° C. and at a pH of about5.7 to about 6.5, to an OD₆₀₀ of about 80 to about 160; and inducing thehost cell at a concentration of about 0.08 mM to about 0.4 mM IPTG;lysing the host cell and centrifuging it to produce the pellet fraction;wherein soluble, active, or soluble and active recombinant interferonprotein can be obtained by extracting the pellet fraction undernon-denaturing conditions without a subsequent renaturing or refoldingstep.
 28. The method of claim 1, further comprising measuring theactivity of the recombinant protein, wherein about 75% to about 100% ofthe recombinant protein present in the extract supernatant is determinedto be active when compared with the amount of active protein in astandard sample, wherein the same amount of protein from each sample isused in the assay.